Sandbox Reserved 1101: Difference between revisions

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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 fibres 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.

'''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 '''fibres''' 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.

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.

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.

==Generalities on fiber assembly of dragline silks==

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.

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'''.

==Overall structure of Major Ampullate Spidroin==

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 (Tokareva, et al., 2013). In Nephila clavipes, there are two distinct MaSp1 genes ; MaSp1A and MaSp1B (Gaines and Marcotte, 2008).

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 (Tokareva, et al., 2013). In ''Nephila clavipes'', there are two distinct MaSp1 genes ; MaSp1A and MaSp1B (Gaines and Marcotte, 2008).

The MaSps are between 250 to 350 kDa 2. They are divided into three parts : C-terminal domain (CTD), repeat domain (RD), and N-terminal domain (NTD).

The MaSps are between 250 to 350 kDa 2. They are divided into three parts : '''C-terminal domain (CTD)''', '''repeat domain (RD)''', and '''N-terminal domain (NTD)'''.

Repeat domain (RD):

*'''Repeat domain (RD)''':

The MaSp sequence corresponds to more than 90% of RD (Romer and Scheibel, 2008). 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 2.

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 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).

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).

C-terminal domain (CTD):

*'''C-terminal domain (CTD)''':

The CTD is a non-repetitive sequence of about 150 amino acids 2. The sequence identity, secondary structure and overall physical properties of CTD is highly conserved across spider species 2. 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.

The CTD is a '''non-repetitive sequence''' of about 150 amino acids 2. The sequence identity, secondary structure and overall physical properties of CTD is highly conserved across spider species 2. 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.

The CTD also plays a role in the change of MaSps solubility according to its localisation in the gland 2. 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.

N-terminal domain (NTD):

*'''N-terminal domain (NTD)''':

This domain is the most highly conserved domain. NTD dimerises in the duct upon conditions change, which connects the MaSps to form fibers.

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 ----
-IZ2 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 fibres 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.
+'''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 '''fibres''' 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.
-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.
+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.
 ==Generalities on fiber assembly of dragline silks==
-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.
+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'''.
 ==Overall structure of Major Ampullate Spidroin==
-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 (Tokareva, et al., 2013). In Nephila clavipes, there are two distinct MaSp1 genes ; MaSp1A and MaSp1B (Gaines and Marcotte, 2008).
+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 (Tokareva, et al., 2013). In ''Nephila clavipes'', there are two distinct MaSp1 genes ; MaSp1A and MaSp1B (Gaines and Marcotte, 2008).
-The MaSps are between 250 to 350 kDa 2. They are divided into three parts : C-terminal domain (CTD), repeat domain (RD), and N-terminal domain (NTD).
+The MaSps are between 250 to 350 kDa 2. They are divided into three parts : '''C-terminal domain (CTD)''', '''repeat domain (RD)''', and '''N-terminal domain (NTD)'''.
-Repeat domain (RD):
+*'''Repeat domain (RD)''':
 The MaSp sequence corresponds to more than 90% of RD (Romer and Scheibel, 2008). 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 2.
-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 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).
+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).
-C-terminal domain (CTD):
+*'''C-terminal domain (CTD)''':
-The CTD is a non-repetitive sequence of about 150 amino acids 2. The sequence identity, secondary structure and overall physical properties of CTD is highly conserved across spider species 2. 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.
+The CTD is a '''non-repetitive sequence''' of about 150 amino acids 2. The sequence identity, secondary structure and overall physical properties of CTD is highly conserved across spider species 2. 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.
 The CTD also plays a role in the change of MaSps solubility according to its localisation in the gland 2. 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.
-N-terminal domain (NTD):
+*'''N-terminal domain (NTD)''':
 This domain is the most highly conserved domain. NTD dimerises in the duct upon conditions change, which connects the MaSps to form fibers.