Sandbox Reserved 1101: Difference between revisions

== Dimerization of the spidroin by the NTD domain ==

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.  

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.

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.  

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.   

Residues T47B, M55B and K54B are more buried at the dimer interface creating specific contacts.  

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.  

K54B engage in a unique hydrogen bond to T43A and electrostatic interaction with D46A.  

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.