Sandbox Reserved 1101: Difference between revisions
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==Overall structure of Major Ampullate Spidroin== | ==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 | 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>. | ||
The MaSps are between 250 to 350 kDa | 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)'''. | ||
*'''Repeat domain (RD)''': | *'''Repeat domain (RD)''': | ||
The MaSp sequence corresponds to more than 90% of RD | 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 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 [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). | 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). | ||
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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. | 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. | ||
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. | 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. | ||
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 | 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"/>. | ||
