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https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Morgane+Bourgeois Proteopedia - User contributions [en] 2026-10-02T21:57:53Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3144149 Sandbox Reserved 1101 2020-01-17T14:57:53Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''N-terminal domain of Major-ampullate Spidroin protein'''<br /> ----<br /> <br /> '''5IZ2''' is the '''N-terminal domain (NTD)''' of a spider protein called '''Major ampullate Spidroin 1A (MaSp1A)''', coming from the ''Nephila Clavipes'' species. This protein is a component of dragline silk produced in the major ampullate gland of spiders<ref name="Atkison"/>. The NTD domain of MaSp1A plays a major role in their combination during silk production <ref name="Atkison"/>. Indeed, thanks to the NTD dimerisation, two MaSps can be connected, leading to the formation of '''fibers''' with exceptional physical and biochemical qualities <ref name="José">José Roberto Aparecido dos Santos-Pinto, Helen Andrade Arcuri, Helga Priewalder, Heliana Clara Salles, Mario Sergio Palma and Gert Lubec, 2015. Structural Model for the Spider Silk Protein Spidroin‑1, Journal of Proteome research, 14, p.3859-3870.</ref>. It is of biotechnological interest to deeply understand the NTD dimerisation mechanism for the production of artificial spider silk, which can lead to innovative biomaterials.<br /> The study of the ''N. Clavipes'' NTD permits to compare its structure with other species thus to provide new insights into the mechanism of NTD dimerization. Moreover, silks produced from different spider breeds vary in physical properties such as toughness and elasticity. In this way, studying diverse species would allow to '''optimize artificial silk''' for different applications.<br /> <br /> <br /> <br /> ==Generalities on fiber assembly of dragline silks==<br /> <br /> The process of the dragline fiber formation is the connection of '''soluble MaSp''' proteins into '''insoluble fibers'''. Indeed, MaSps are firstly secreted and stored in soluble form in the tail of the major ampullate gland which is located in the spider’s abdomen. On demand, they pass through the narrow duct where they experience mechanical and chemical forces that convert them into fibers. Actually, they deal with a pH dropping, an alteration of ion concentrations and oxidation conditions, which occur gradually along the duct. These changes promote the connection of MaSps extremities (i.e. homo-dimerisation of C- and N-terminal domains) to form fibers. Finally, through flow rate and mechanical forces experienced in the duct, the fibers will agglomerate to create the '''dragline silk'''.<br /> <br /> <br /> <br /> ==Overall structure of Major Ampullate Spidroin==<br /> <br /> The dragline fiber is mainly composed of proteins termed Major ampullate Spidroin 1 and Major ampullate Spidroin 2 (MaSp1 and MaSp2). MaSp1 is found in both the core and periphery of the fiber, while MaSp2 is only assembled in the core <ref name="Cadle"/>. In ''Nephila clavipes'', there are two distinct MaSp1 genes ; MaSp1A and MaSp1B <ref>PMID:18828837</ref>. <br /> The MaSps are between 250 to 350 kDa <ref name="Cadle"/>. They are divided into three parts : '''C-terminal domain (CTD)''', '''repeat domain (RD)''', and '''N-terminal domain (NTD)'''. <br /> <br /> <br /> *'''Repeat domain (RD)''':<br /> The MaSp sequence corresponds to more than 90% of RD <ref>PMID:19221522</ref>. The RD is a long, flexible, highly repetitive central domain. It varies greatly between the types of silks, which makes it responsible for their different properties <ref name="Cadle"/>.<br /> MaSp1 contains poly-alanine (A)n motifs at the end of a repeat, as well as GA and GGX motifs where X is often A, Y, L, or Q (Gatesy et al., 2001; Hu et al., 2006; Keten and Buehler, 2010; Xu and Lewis, 1990). The poly-alanine motifs, usually present at the end of a repeat, form [https://en.wikipedia.org/wiki/Beta_sheet_ β-sheets] in the duct due to mechanical forces. The β-sheets will then line up in parallel, leading to the aggregation of the fibers. The GGX motifs form an amorphous matrix that connects the crystalline regions (Hayashi et al., 1999; Scheibel, 2004).<br /> <br /> <br /> *'''C-terminal domain (CTD)''':<br /> The CTD is a '''non-repetitive sequence''' of about 150 amino acids <ref name="Cadle"/>. The sequence identity, secondary structure and overall physical properties of CTD is highly conserved across spider species <ref name="Cadle"/>. Its structure forms a bundle of five parallel α-helices. A single '''cysteine residue''' in the middle of its sequence is highly conserved and is responsible for the CTD homo-dimerisation. In other words, it allows the covalent connection between two CTDs through disulfide bond linkage. <br /> The CTD also plays a role in the change of MaSps solubility according to its localisation in the gland <ref name="Cadle"/>. Indeed, it presents a high number of charged and polar amino acids present in its sequence. In this way, when the pH is neutral in the ampullate, the hydrophobic residues are buried within the core and the hydrophilic residues are exposed. This permits to keep the MaSps soluble, preventing early fiber aggregation. On the contrary, when the CTDs are in the duct with lower pH, the acidic residues switch from a negative to a neutral charge. This leads to an increase of hydrophobic interactions that help with the formation of β-sheets and thus MaSps precipitation.<br /> <br /> <br /> *'''N-terminal domain (NTD)''': <br /> This domain is the most highly conserved domain. NTD dimerises in the duct upon conditions change, which connects the MaSps to form fibers.<br /> <br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 parallel [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>)<ref name="Cadle"/>. <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process<ref name="Atkison"/>.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are '''clusters of acidic residus''' (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and '''clusters of basic residus''' (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a '''dipole moment''', which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues<ref name="Atkison"/>. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a '''different helices arrangement'''. So they do not completely overlap. This allows the creation of '''new intermolecular contact networks'''. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet<ref name="Atkison"/>.<br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a '''rearrangement of the five-helix bundle''' during the monomer to dimer transition. An '''acidification''' along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit '''selects a partner''' with a complementary binding interface.<ref name="Atkison"/> When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to '''salt bridges formation'''.<ref name="Cadle">Cadle KA, 2016. “The Role the N-terminal Domain Plays in Spidroin Assembly”, All Dissertations. 2296 https://tigerprints.clemson.edu/all_dissertations/2296/?utm_source=tigerprints.clemson.edu%252Fall_dissertations%252F2296&utm_medium=PDF&utm_campaign=PDFCoverPages.</ref> Moreover, dimerization is really '''triggered and stabilized''' by '''protonation''' of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The '''plasticity''' of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <ref name="Atkison"/><br /> <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the '''intramolecular handshake interaction'''. The '''asymmetric nature''' and the '''difference of topology''' of the subunits allow the formation of '''salt bridges'''. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that '''Asp39 is essential''' for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits '''alignment better'''. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the '''variability in the interactions''' that take place to Lys65. This variability provides a '''mechanism for plasticity''' in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291 no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These '''asymmetric contacts''' play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than '''38,5%''' of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the '''specific angles''' at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are '''more buried''' at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in '''Van Der Waals interactions''' with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a '''Van Der Waals interaction''' of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a '''unique hydrogen bond''' to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are '''less buried''' than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into '''hydrophobic pockets''' formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the '''pH-dependent NTD dimerization mechanism''', a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at '''pH 6,1''''. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that '''short-range asymmetric salt bridges''' between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the '''handshake interaction''' and also the '''protonation of Glu84''', which must be preceded by protonation of Glu79 and Glu119. Similarly, the '''protonation of Asp17 and Asp53''' plays also a key role in the mechanism of NTD dimerization <ref name="Atkison"/>. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct <ref name="Cadle"/>.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3144148 Sandbox Reserved 1101 2020-01-17T14:57:19Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''N-terminal domain of Major-ampullate Spidroin protein'''<br /> ----<br /> <br /> '''5IZ2''' is the '''N-terminal domain (NTD)''' of a spider protein called '''Major ampullate Spidroin 1A (MaSp1A)''', coming from the ''Nephila Clavipes'' species. This protein is a component of dragline silk produced in the major ampullate gland of spiders<ref name="Atkison"/>. The NTD domain of MaSp1A plays a major role in their combination during silk production <ref name="Atkison"/>. Indeed, thanks to the NTD dimerisation, two MaSps can be connected, leading to the formation of '''fibers''' with exceptional physical and biochemical qualities <ref name="José">José Roberto Aparecido dos Santos-Pinto, Helen Andrade Arcuri, Helga Priewalder, Heliana Clara Salles, Mario Sergio Palma and Gert Lubec, 2015. Structural Model for the Spider Silk Protein Spidroin‑1, Journal of Proteome research, 14, p.3859-3870.</ref>. It is of biotechnological interest to deeply understand the NTD dimerisation mechanism for the production of artificial spider silk, which can lead to innovative biomaterials.<br /> The study of the ''N. Clavipes'' NTD permits to compare its structure with other species thus to provide new insights into the mechanism of NTD dimerization. Moreover, silks produced from different spider breeds vary in physical properties such as toughness and elasticity. In this way, studying diverse species would allow to '''optimize artificial silk''' for different applications.<br /> <br /> <br /> <br /> ==Generalities on fiber assembly of dragline silks==<br /> <br /> The process of the dragline fiber formation is the connection of '''soluble MaSp''' proteins into '''insoluble fibers'''. Indeed, MaSps are firstly secreted and stored in soluble form in the tail of the major ampullate gland which is located in the spider’s abdomen. On demand, they pass through the narrow duct where they experience mechanical and chemical forces that convert them into fibers. Actually, they deal with a pH dropping, an alteration of ion concentrations and oxidation conditions, which occur gradually along the duct. These changes promote the connection of MaSps extremities (i.e. homo-dimerisation of C- and N-terminal domains) to form fibers. Finally, through flow rate and mechanical forces experienced in the duct, the fibers will agglomerate to create the '''dragline silk'''.<br /> <br /> <br /> <br /> ==Overall structure of Major Ampullate Spidroin==<br /> <br /> The dragline fiber is mainly composed of proteins termed Major ampullate Spidroin 1 and Major ampullate Spidroin 2 (MaSp1 and MaSp2). MaSp1 is found in both the core and periphery of the fiber, while MaSp2 is only assembled in the core <ref name="Cadle"/>. In ''Nephila clavipes'', there are two distinct MaSp1 genes ; MaSp1A and MaSp1B <ref>PMID:18828837</ref>. <br /> The MaSps are between 250 to 350 kDa <ref name="Cadle"/>. They are divided into three parts : '''C-terminal domain (CTD)''', '''repeat domain (RD)''', and '''N-terminal domain (NTD)'''. <br /> <br /> <br /> *'''Repeat domain (RD)''':<br /> The MaSp sequence corresponds to more than 90% of RD <ref>PMID:19221522</ref>. The RD is a long, flexible, highly repetitive central domain. It varies greatly between the types of silks, which makes it responsible for their different properties <ref name="Cadle"/>.<br /> MaSp1 contains poly-alanine (A)n motifs at the end of a repeat, as well as GA and GGX motifs where X is often A, Y, L, or Q (Gatesy et al., 2001; Hu et al., 2006; Keten and Buehler, 2010; Xu and Lewis, 1990). The poly-alanine motifs, usually present at the end of a repeat, form [https://en.wikipedia.org/wiki/Beta_sheet_ β-sheets] in the duct due to mechanical forces. The β-sheets will then line up in parallel, leading to the aggregation of the fibers. The GGX motifs form an amorphous matrix that connects the crystalline regions (Hayashi et al., 1999; Scheibel, 2004).<br /> <br /> <br /> *'''C-terminal domain (CTD)''':<br /> The CTD is a '''non-repetitive sequence''' of about 150 amino acids <ref name="Cadle"/>. The sequence identity, secondary structure and overall physical properties of CTD is highly conserved across spider species <ref name="Cadle"/>. Its structure forms a bundle of five parallel α-helices. A single '''cysteine residue''' in the middle of its sequence is highly conserved and is responsible for the CTD homo-dimerisation. In other words, it allows the covalent connection between two CTDs through disulfide bond linkage. <br /> The CTD also plays a role in the change of MaSps solubility according to its localisation in the gland <ref name="Cadle"/>. Indeed, it presents a high number of charged and polar amino acids present in its sequence. In this way, when the pH is neutral in the ampullate, the hydrophobic residues are buried within the core and the hydrophilic residues are exposed. This permits to keep the MaSps soluble, preventing early fiber aggregation. On the contrary, when the CTDs are in the duct with lower pH, the acidic residues switch from a negative to a neutral charge. This leads to an increase of hydrophobic interactions that help with the formation of β-sheets and thus MaSps precipitation.<br /> <br /> <br /> *'''N-terminal domain (NTD)''': <br /> This domain is the most highly conserved domain. NTD dimerises in the duct upon conditions change, which connects the MaSps to form fibers.<br /> <br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 parallel [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>)<ref name="Cadle"/>. <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process<ref name="Atkison"/>.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are '''clusters of acidic residus''' (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and '''clusters of basic residus''' (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a '''dipole moment''', which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues<ref name="Atkison"/>. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a '''different helices arrangement'''. So they do not completely overlap. This allows the creation of '''new intermolecular contact networks'''. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet<ref name="Atkison"/>.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a '''rearrangement of the five-helix bundle''' during the monomer to dimer transition. An '''acidification''' along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit '''selects a partner''' with a complementary binding interface.<ref name="Atkison"/> When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to '''salt bridges formation'''.<ref name="Cadle">Cadle KA, 2016. “The Role the N-terminal Domain Plays in Spidroin Assembly”, All Dissertations. 2296 https://tigerprints.clemson.edu/all_dissertations/2296/?utm_source=tigerprints.clemson.edu%252Fall_dissertations%252F2296&utm_medium=PDF&utm_campaign=PDFCoverPages.</ref> Moreover, dimerization is really '''triggered and stabilized''' by '''protonation''' of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The '''plasticity''' of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <ref name="Atkison"/><br /> <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the '''intramolecular handshake interaction'''. The '''asymmetric nature''' and the '''difference of topology''' of the subunits allow the formation of '''salt bridges'''. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that '''Asp39 is essential''' for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits '''alignment better'''. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the '''variability in the interactions''' that take place to Lys65. This variability provides a '''mechanism for plasticity''' in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291 no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These '''asymmetric contacts''' play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than '''38,5%''' of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the '''specific angles''' at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are '''more buried''' at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in '''Van Der Waals interactions''' with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a '''Van Der Waals interaction''' of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a '''unique hydrogen bond''' to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are '''less buried''' than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into '''hydrophobic pockets''' formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the '''pH-dependent NTD dimerization mechanism''', a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at '''pH 6,1''''. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that '''short-range asymmetric salt bridges''' between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the '''handshake interaction''' and also the '''protonation of Glu84''', which must be preceded by protonation of Glu79 and Glu119. Similarly, the '''protonation of Asp17 and Asp53''' plays also a key role in the mechanism of NTD dimerization <ref name="Atkison"/>. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct <ref name="Cadle"/>.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143702 Sandbox Reserved 1101 2020-01-16T13:07:11Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the '''dragline silk'''. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the '''dimerization of spidroins'''. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are '''clusters of acidic residus''' (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and '''clusters of basic residus''' (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a '''dipole moment''', which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a '''different helices arrangement'''. So they do not completely overlap. This allows the creation of '''new intermolecular contact networks'''. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a '''rearrangement of the five-helix bundle''' during the monomer to dimer transition. An '''acidification''' along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit '''selects a partner''' with a complementary binding interface.<ref name="Atkison"/> When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to '''salt bridges formation'''.<ref name="Cadle">Cadle KA, 2016. “The Role the N-terminal Domain Plays in Spidroin Assembly”, All Dissertations. 2296 https://tigerprints.clemson.edu/all_dissertations/2296/?utm_source=tigerprints.clemson.edu%252Fall_dissertations%252F2296&utm_medium=PDF&utm_campaign=PDFCoverPages.</ref> Moreover, dimerization is really '''triggered and stabilized''' by '''protonation''' of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The '''plasticity''' of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <ref name="Atkison"/><br /> <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the '''intramolecular handshake interaction'''. The '''asymmetric nature''' and the '''difference of topology''' of the subunits allow the formation of '''salt bridges'''. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that '''Asp39 is essential''' for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits '''alignment better'''. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the '''variability in the interactions''' that take place to Lys65. This variability provides a '''mechanism for plasticity''' in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291 no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These '''asymmetric contacts''' play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than '''38,5%''' of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the '''specific angles''' at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are '''more buried''' at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in '''Van Der Waals interactions''' with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a '''Van Der Waals interaction''' of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a '''unique hydrogen bond''' to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are '''less buried''' than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into '''hydrophobic pockets''' formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the '''pH-dependent NTD dimerization mechanism''', a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at '''pH 6,1''''. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that '''short-range asymmetric salt bridges''' between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the '''handshake interaction''' and also the '''protonation of Glu84''', which must be preceded by protonation of Glu79 and Glu119. Similarly, the '''protonation of Asp17 and Asp53''' plays also a key role in the mechanism of NTD dimerization. <ref name="Atkison"/>These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name="Cadle"/><br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143495 Sandbox Reserved 1101 2020-01-15T19:41:18Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the '''dragline silk'''. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the '''dimerization of spidroins'''. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are '''clusters of acidic residus''' (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and '''clusters of basic residus''' (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a '''dipole moment''', which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a '''different helices arrangement'''. So they do not completely overlap. This allows the creation of '''new intermolecular contact networks'''. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a '''rearrangement of the five-helix bundle''' during the monomer to dimer transition. An '''acidification''' along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit '''selects a partner''' with a complementary binding interface.<ref name="Atkison"/> When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to '''salt bridges formation'''.<ref name="Cadle">Cadle KA, 2016. “The Role the N-terminal Domain Plays in Spidroin Assembly”, All Dissertations. 2296 https://tigerprints.clemson.edu/all_dissertations/2296/?utm_source=tigerprints.clemson.edu%252Fall_dissertations%252F2296&utm_medium=PDF&utm_campaign=PDFCoverPages.</ref> Moreover, dimerization is really '''triggered and stabilized''' by '''protonation''' of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The '''plasticity''' of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <ref name="Atkison"/><br /> <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the '''intramolecular handshake interaction'''. The '''asymmetric nature''' and the '''difference of topology''' of the subunits allow the formation of '''salt bridges'''. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that '''Asp39 is essential''' for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits '''alignment better'''. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the '''variability in the interactions''' that take place to Lys65. This variability provides a '''mechanism for plasticity''' in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These '''asymmetric contacts''' play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than '''38,5%''' of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the '''specific angles''' at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are '''more buried''' at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in '''Van Der Waals interactions''' with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a '''Van Der Waals interaction''' of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a '''unique hydrogen bond''' to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are '''less buried''' than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into '''hydrophobic pockets''' formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the '''pH-dependent NTD dimerization mechanism''', a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at '''pH 6,1''''. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that '''short-range asymmetric salt bridges''' between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the '''handshake interaction''' and also the '''protonation of Glu84''', which must be preceded by protonation of Glu79 and Glu119. Similarly, the '''protonation of Asp17 and Asp53''' plays also a key role in the mechanism of NTD dimerization. <ref name="Atkison"/>These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name="Cadle"/><br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143488 Sandbox Reserved 1101 2020-01-15T19:31:09Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the '''dragline silk'''. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the '''dimerization of spidroins'''. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are '''clusters of acidic residus''' (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and '''clusters of basic residus''' (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a '''dipole moment''', which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a '''different helices arrangement'''. So they do not completely overlap. This allows the creation of '''new intermolecular contact networks'''. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface.<ref name="Atkison"/> When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation.<ref name="Cadle">Cadle KA, 2016. “The Role the N-terminal Domain Plays in Spidroin Assembly”, All Dissertations. 2296 https://tigerprints.clemson.edu/all_dissertations/2296/?utm_source=tigerprints.clemson.edu%252Fall_dissertations%252F2296&utm_medium=PDF&utm_campaign=PDFCoverPages.</ref> Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <ref name="Atkison"/><br /> <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. <ref name="Atkison"/>These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name="Cadle"/><br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143484 Sandbox Reserved 1101 2020-01-15T19:27:48Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface.<ref name="Atkison"/> When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation.<ref name="Cadle">Cadle KA, 2016. “The Role the N-terminal Domain Plays in Spidroin Assembly”, All Dissertations. 2296 https://tigerprints.clemson.edu/all_dissertations/2296/?utm_source=tigerprints.clemson.edu%252Fall_dissertations%252F2296&utm_medium=PDF&utm_campaign=PDFCoverPages.</ref> Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <ref name="Atkison"/><br /> <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. <ref name="Atkison"/>These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name="Cadle"/><br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143480 Sandbox Reserved 1101 2020-01-15T19:26:22Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface.<ref name="Atkison"/> When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation.<ref name="Cadle">Cadle KA, 2016. “The Role the N-terminal Domain Plays in Spidroin Assembly”, All Dissertations. 2296 https://tigerprints.clemson.edu/all_dissertations/2296/?utm_source=tigerprints.clemson.edu%252Fall_dissertations%252F2296&utm_medium=PDF&utm_campaign=PDFCoverPages.</ref><br /> Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <ref name="Atkison"/><br /> <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name="Atkison"/><br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143472 Sandbox Reserved 1101 2020-01-15T19:17:33Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref name="Atkison"/><br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface.<ref name="Atkison"/><br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref name="Atkison"/> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref name="Atkison"/><br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref name="Atkison"/><br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name="Atkison"/><br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143470 Sandbox Reserved 1101 2020-01-15T19:16:38Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref name="Atkison">Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref name="Atkison"/><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143466 Sandbox Reserved 1101 2020-01-15T19:12:31Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref><br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143463 Sandbox Reserved 1101 2020-01-15T19:10:30Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143459 Sandbox Reserved 1101 2020-01-15T19:09:24Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer <ref>Atkison JH, Parnham S, Marcotte WR, Jr., and Olsen SK, 2016. Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface, The Journal of Biological Chemistry, vol.291n no.36, p.19006-19017.</ref> <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143375 Sandbox Reserved 1101 2020-01-15T15:20:24Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilizes even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6 Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1 Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> <br /> ::*''Van der Waals''<br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143370 Sandbox Reserved 1101 2020-01-15T15:10:45Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and <scene name='82/829354/F127/1'>F127A</scene> also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143368 Sandbox Reserved 1101 2020-01-15T15:05:48Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, <scene name='82/829354/M126/1'>M126A</scene> and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143356 Sandbox Reserved 1101 2020-01-15T14:26:31Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> ======''Principal interactions''======<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> ======''Secondary interactions'' ======<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> <br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143352 Sandbox Reserved 1101 2020-01-15T14:08:12Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a <scene name='82/829354/Chain_z/1'>chain Z</scene> composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143351 Sandbox Reserved 1101 2020-01-15T14:05:32Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (<scene name='82/829354/A/1'>H1 to H5</scene>). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143348 Sandbox Reserved 1101 2020-01-15T14:01:43Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> <br /> <br /> <br /> ==Global structure of the ''N. Clavipes'' Spidroin-1==<br /> <br /> <br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> <Structure load='5iz2' size='350' frame='true' align='right' caption='NTD monomer' scene='Insert optional scene name here' /><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> ==Dimerization of the spidroin by the NTD domain==<br /> <br /> <br /> <br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> <StructureSection load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143346 Sandbox Reserved 1101 2020-01-15T13:53:50Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> <StructureSection load='5iz2' size='350' side='right' caption='NTD monomer' scene=''><br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). <br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks. There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='N-Terminale domain dimer' scene='Insert optional scene name here' /><br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143338 Sandbox Reserved 1101 2020-01-15T13:19:13Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp39 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143337 Sandbox Reserved 1101 2020-01-15T13:18:32Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. In subunits H2A and H2B, T47A and A51B engage in a Van Der Waals interaction of 4,1 Å, and that contribute to the plasticity of the dimer interface. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> ::*''Hydrophobic pockets''<br /> Then, in subunits H5A and H5B, M126A and F127A also buried at the dimer interface, insert into hydrophobic pockets formed by S122B, L123B and M71B, S75B, E119B and I120B respectively. <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143335 Sandbox Reserved 1101 2020-01-15T13:16:02Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, <scene name='82/829354/Asp40b_glu84b/1'>Asp40 and Glu84 of subunit B</scene> engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143333 Sandbox Reserved 1101 2020-01-15T13:07:56Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> * ''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/2'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in ''N.clavipes''. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between <scene name='82/829354/Asp17a-asp53a_interaction/1'>Asp17 and Asp53 in subunit A</scene>. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> * ''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in ''N. clavipes'' several other novel interactions occur. For example, in comparison with the ''Euprosthenops australis'' NTD, ''N. clavipes'' NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ::*''Van der Waals''<br /> <scene name='82/829354/T47b-i48a-a51a-l69a/1'>T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues</scene>. Also, <scene name='82/829354/M55b-d40a-t43a/2'>M55B is commited in Van Der Waals interactions with D40A and T43A</scene>. <br /> <br /> ::*''Hydrogen bonds and electrostatic interactions''<br /> K54B engage in a unique hydrogen bond to <scene name='82/829354/K54b-t43a/1'>T43A</scene> and electrostatic interaction with <scene name='82/829354/K54b-d46a/1'>D46A</scene>. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The ''N. clavipes'' NTD contains a single tryptophan (Trp10) near the N-terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143224 Sandbox Reserved 1101 2020-01-14T20:06:54Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> *''Principal interactions''<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/1'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in "N.clavipes". These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> *''Secondary interactions'' <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in N. clavipes several other novel interactions occur. For example, in comparison with the Euprosthenops australis NTD, N. clavipes NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> **Van der Waals<br /> T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues. Also, M55B is commited in Van Der Waals interactions with D40A and T43A. <br /> <br /> **Hydrogen bonds and electrostatic interactions<br /> K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The N. clavipes NTD contains a single tryptophan (Trp10) near the N terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143223 Sandbox Reserved 1101 2020-01-14T20:05:20Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ====Interactions====<br /> <br /> *Principal interactions<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/1'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in "N.clavipes". These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> *Secondary interactions <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in N. clavipes several other novel interactions occur. For example, in comparison with the Euprosthenops australis NTD, N. clavipes NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> **Van der Waals<br /> T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues. Also, M55B is commited in Van Der Waals interactions with D40A and T43A. <br /> <br /> **Hydrogen bonds and electrostatic interactions<br /> K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> <br /> ====pH-dependent mechanism====<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The N. clavipes NTD contains a single tryptophan (Trp10) near the N terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143222 Sandbox Reserved 1101 2020-01-14T20:04:52Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ====Conformational change of the five-helix bundle====<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ===Interactions===<br /> <br /> *Principal interactions<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/1'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in "N.clavipes". These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> *Secondary interactions <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in N. clavipes several other novel interactions occur. For example, in comparison with the Euprosthenops australis NTD, N. clavipes NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> **Van der Waals<br /> T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues. Also, M55B is commited in Van Der Waals interactions with D40A and T43A. <br /> <br /> **Hydrogen bonds and electrostatic interactions<br /> K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> <br /> ===pH-dependent mechanism===<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The N. clavipes NTD contains a single tryptophan (Trp10) near the N terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143221 Sandbox Reserved 1101 2020-01-14T20:03:43Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> ===Conformational change of the five-helix bundle===<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> ===Interactions===<br /> <br /> *Principal interactions<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/1'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in "N.clavipes". These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> *Secondary interactions <br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in N. clavipes several other novel interactions occur. For example, in comparison with the Euprosthenops australis NTD, N. clavipes NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> **Van der Waals<br /> T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues. Also, M55B is commited in Van Der Waals interactions with D40A and T43A. <br /> <br /> **Hydrogen bonds and electrostatic interactions<br /> K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> <br /> ===pH-dependent mechanism===<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The N. clavipes NTD contains a single tryptophan (Trp10) near the N terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143218 Sandbox Reserved 1101 2020-01-14T20:00:44Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> * Conformational change of the five-helix bundle<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> *Interactions<br /> <br /> **<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/1'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in "N.clavipes". These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> **Secondary interactions<br /> <br /> These asymmetric contacts play a well-defined role in dimer formation in many species of spiders but in N. clavipes several other novel interactions occur. For example, in comparison with the Euprosthenops australis NTD, N. clavipes NTD engage more than 38,5% of novel interactions. These ones result from the distinct topology of the three helices (H2, H3 and H5) compared to other species. Indeed, the specific angles at which the H2, H3 and H5 helices cross their counterparts in the asymmetric interface allow the correct positioning of residues and the establishment of these interactions. <br /> Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts. <br /> <br /> ***Van der Waals<br /> T47B engage in Van Der Waals contacts with I48A, A51A and L69A residues. Also, M55B is commited in Van Der Waals interactions with D40A and T43A. <br /> <br /> ***Hydrogen bonds and electrostatic interactions<br /> K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A. <br /> <br /> On the other side on the dimer interface, there are also other specific contacts but distinct due to the different topology. But residues T47A, K54A and M55A are less buried than their counterparts in subunit A in particularly K54A which doesn’t engage any interaction. <br /> <br /> <br /> *pH-dependent mechanism<br /> <br /> In order to observe the pH-dependent NTD dimerization mechanism, a tryptophan fluorescence assay was used. The N. clavipes NTD contains a single tryptophan (Trp10) near the N terminus. During the transition from the NTD monomer to the NTD dimer, a conformational change occurs for Trp10 that increases its solvent exposure. As a consequence, a quenching of its fluorescence emission is observed. The transition from the NTD monomer to the NTD dimer occurs at pH 6,1. At pH above 6,1, NTD is in the form of monomer and the formation of dimer occurs after pH 6,1. <br /> Mutations in residues Asp40, Lys65 involved in salt bridges result in decrease in dimer stability. This assay shows that short-range asymmetric salt bridges between Asp39, Asp40 and Lys65 are essential to the NTD dimerization. <br /> Next, a mutation of residue Glu84 completely destabilize the dimer formation, that shows the importance of the handshake interaction and also the protonation of Glu84, which must be preceded by protonation of Glu79 and Glu119. Similarly, the protonation of Asp17 and Asp53 plays also a key role in the mechanism of NTD dimerization. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<br /> <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3143216 Sandbox Reserved 1101 2020-01-14T19:57:20Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called [https://en.wikipedia.org/wiki/Spidroin_ spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important since it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, two spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 [https://en.wikipedia.org/wiki/Alpha_helix_ α-helix] (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> * Conformational change of the five-helix bundle<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle during the monomer to dimer transition. An acidification along the spinning duct results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> *Interactions<br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges. <scene name='82/829354/Lys_65-asp39_interaction/1'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, <scene name='82/829354/Lys_65b-asp40a_interaction/1'>Asp40 of subunit A and Lys65 of subunit B</scene> engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders and seems to be also important in "N.clavipes". These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <scene name='82/829354/Asp40-glu84/3'>intramolecular handshake interaction between Asp40 and Glu84</scene><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142885 Sandbox Reserved 1101 2020-01-13T15:02:38Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : Crystal structure of the ''N. Clavipes'' spidroin NTD'''<br /> ----<br /> <br /> 5IZ2 is the '''NTD domain''' of a protein called '''spidroin'''[https://en.wikipedia.org/wiki/Spidroin]. This protein is a component of the dragline silk. There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important because it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, 2 spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD (N-Terminal Domain) is composed of 5 α-helices (H1 to H5). There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. In addition to this, the subunits A and B are organized antiparallel, which allows an access to charges poles. <br /> The charged residues (the acidic and basic ones) are responsible for creating a dipole moment, which therefore implies a non-uniform charge arrangement within the subunits. This is important for the dimerization process, that is why they are highly conserved residues. <br /> <br /> Compared with spidroin of other species of spider, the 2 subunits (A and B) of the dimerized NTD of the spidroin produced by ''N. Clavipes'' are slightly different, due to a different helices arrangement. So they do not completely overlap. This allows the creation of new intermolecular contact networks.<br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle occurs during the monomer to dimer transition. An acidification of the medium results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. <scene name='82/829354/Lys_65-asp39_interaction/1'>Lys65 of subunit A and Asp39 of subunit B</scene> engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, Asp40 of subunit A and Lys65 of subunit B engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. <br /> <br /> The structure of ''N. clavipes'' dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders (16). The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges between Asp39 and Lys65 and between Asp40 and Lys65. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> Another intramolecular handshake interaction occurs also between Asp17 and Asp53 in subunit A. This interaction doesn’t exist in subunit B because of the orientation of subunit A with respect to subunit B, Asp17 and Asp53 are too far away in order to engage this interaction. <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <scene name='82/829354/Asp40-glu84/3'>intramolecular handshake interaction between Asp40 and Glu84</scene><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142812 Sandbox Reserved 1101 2020-01-12T20:42:16Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : Crystal structure of the ''N. Clavipes'' spidroin NTD'''<br /> <br /> 5IZ2 is the NTD domain of a protein called spidroin[https://en.wikipedia.org/wiki/Spidroin]. This protein is a component of the dragline silk.<br /> There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important because it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, 2 spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD is composed of 2 subunits, A and B, each of them composed of 5 α-helices. There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. And, as the subunits A and B are organized antiparallel, it allows an access to charges poles. <br /> <br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle occurs during the monomer to dimer transition. An acidification of the medium results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> Different types of interactions occur between specific residues during the NTD dimerization. Asp40, Lys65, Asp39 and Glu84 residues have been identified as being particularly important.<br /> In one side, Asp40 and Glu84 of subunit A engage in the intramolecular handshake interaction. Lys65 of subunit A and Asp39 of subunit B engage in a short-range intermolecular salt bridge of 2,6Å. In the other side, Asp40 of subunit A and Lys65 of subunit B engage in a short-range intermolecular salt bridge of 3,1Å. Asp39 is not involved in this part of the dimer. <br /> <br /> The structure of N. clavipes dimer interface differs from those of other species due to the asymmetric nature of the interface and the involvement of Asp39. It has been reported that Asp39 is essential for the NTD dimerization in other species of spiders (16). The asymmetric nature and the difference of topology of the subunits allow the formation of salt bridges between Asp39 and Lys65 and between Asp40 and Lys65. These interactions make subunits alignment better. Acidic residues are conserved around residues Asp96 and Asp40 and this allows the variability in the interactions that take place to Lys65. This variability provides a mechanism for plasticity in the dimer interface allowing the transition from loosely to stably associated dimer. <br /> <br /> <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <scene name='82/829354/Asp40-glu84/3'>intramolecular handshake interaction between Asp40 and Glu84</scene><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142811 Sandbox Reserved 1101 2020-01-12T20:41:24Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> <br /> <br /> '''5IZ2 : Crystal structure of the ''N. Clavipes'' spidroin NTD'''<br /> <br /> 5IZ2 is the NTD domain of a protein called spidroin[https://en.wikipedia.org/wiki/Spidroin]. This protein is a component of the dragline silk.<br /> There are several types of spidroin, and those that form the core of the silk are called '''MaSp1''' (Major ampullate Spidroin-1), which are produced by in the major ampullate gland of spiders. <br /> The NTD domain of these proteins is very important because it plays a major role in the dimerisation of spidroins. Indeed, thanks to the NTD organization, 2 spidroins can be combined, leading to the production of fibres with exceptional physical qualities.<br /> <br /> <br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Global structure of the ''N. Clavipes'' Spidroin-1 ==<br /> <br /> == Monomer structure of the spidroin NTD domain ==<br /> <br /> One monomer of NTD is composed of 2 subunits, A and B, each of them composed of 5 α-helices. There is also a chain Z composed of 3 amino acids (Ser, Tyr, Gly), but it role is not well established yet.<br /> <br /> In each subunit, the orientation of helices 2, 3 and 5 is different from the orientation of helices 1 and 4. Indeed, helices 1 and 4 form the rigid body of the NTD domain, while helices 2, 3 and 5 are involved in intermolecular contacts, so they play an important role in the dimerization process.<br /> <br /> Moreover, at the opposite extremities of each subunits of the monomer there are clusters of acidic residus (Asp36, Asp39, Asp40, Glu79, Asp91) in one part, and clusters of basic residus (Lys54, Arg57, Lys60, Lys64, Lys65) in the other part. So, this create a dipole moment. And, as the subunits A and B are organized antiparallel, it allows an access to charges poles. <br /> <br /> <br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> The dimerization of the spidroin by the NTD domain begins by a rearrangement of the five-helix bundle occurs during the monomer to dimer transition. An acidification of the medium results in a conformational change of the NTD. So, for the NTD dimerization, a lowering of pH from 7 to 6 is important. Then, a subunit selects a partner with a complementary binding interface. When the NTD forms a dimer, its positive and negative poles are opposed, creating an environment conducive to salt bridges formation. Moreover, dimerization is really triggered and stabilized by protonation of some residues. Studies have also shown that a lowering more important of the pH stabilize even more the dimer. The plasticity of the dimer interface could also be a factor of the conformational selection during transition from monomer to dimer or during the transition from loosely to stably dimer. <br /> <br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <scene name='82/829354/Asp40-glu84/3'>intramolecular handshake interaction between Asp40 and Glu84</scene><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> <br /> == Applications in Biotechnology ==<br /> <br /> </StructureSection><br /> <br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142435 Sandbox Reserved 1101 2020-01-10T16:22:58Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <scene name='82/829354/Asp40-glu84/3'>intramolecular handshake interaction between Asp40 and Glu84</scene><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142433 Sandbox Reserved 1101 2020-01-10T16:17:46Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> <scene name='82/829354/Asp40-glu84/1'>intramolecular handshake interaction between Asp40 and Glu84</scene><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox1101&diff=3142429 Sandbox1101 2020-01-10T15:36:08Z

<p>Morgane Bourgeois: New page: ==Your Heading Here (maybe something like 'Structure')== <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''> This is a default text for you...</p> <hr /> <div>==Your Heading Here (maybe something like 'Structure')==<br /> <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''><br /> This is a default text for your page '''Sandbox1101'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Relevance ==<br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142413 Sandbox Reserved 1101 2020-01-09T20:31:37Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142412 Sandbox Reserved 1101 2020-01-09T20:31:05Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''><br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> <Structure load='5IZ2' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Morgane Bourgeois https://proteopedia.org/index.php?title=Sandbox_Reserved_1101&diff=3142411 Sandbox Reserved 1101 2020-01-09T20:29:21Z

<p>Morgane Bourgeois: </p> <hr /> <div>{{Sandbox_ESBS_2019}}<br /> ==Your Heading Here (maybe something like 'Structure')==<br /> <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''><br /> This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br /> You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br /> <br /> == Function ==<br /> <br /> == Disease ==<br /> <br /> == Dimerization of the spidroin by the NTD domain ==<br /> <br /> <br /> <br /> == Structural highlights ==<br /> <br /> This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br /> <br /> </StructureSection><br /> == References ==<br /> <references/></div>

Morgane Bourgeois