Sandbox Reserved 1101: Difference between revisions
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====Conformational change of the five-helix bundle==== | ====Conformational change of the five-helix bundle==== | ||
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> | 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"/> | ||
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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. These protonations are allowed by the lowering of the pH suffered by the NTD during its progression in the spinning duct.<ref name=" | 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"/> | ||
