Date of Award

1-1-2016

Embargo Period

1-1-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Shaun Olsen

Second Advisor

Daniel Bearden

Third Advisor

Joe Blumer

Fourth Advisor

Christoper Davies

Fifth Advisor

Tilman Heise

Abstract

Spider dragline silk is a naturally occurring polymer that has the potential to be used in many biomaterials such as skin graft scaffolds and cartilage repair matrices due to its elasticity, high tensile strength, and biocompatibility. However, natural large scale production of spider silk is unfeasible due to the cannibalistic nature of spiders in captivity. A viable alternative is artificial silk production, which requires an understanding of the in vivo mechanisms spiders use to spin silk. The conversion of highly concentrated proteins, called spidroins, into insoluble fibers requires the dimerization of the spidroin N-terminal domain (NTD). This process is regulated by decreases in both salt concentration and pH; however, the specific mechanisms involved are still not completely known. To gain a more detailed understanding of these processes, I solved the crystal structure of the Nephila clavipes major ampullate spidroin 1A (NcNTD). The structure contains unique intermolecular contacts including key salt bridges that grant a degree of plasticity at the dimer interface. Additionally, I observed a novel intramolecular handshake interaction between highly conserved acidic residues D17 and D53. Interestingly, the D17A mutant favored dimer formation of the NTD; thus, I also solved its crystal structure for comparison with wild type. Additionally, I used NMR-based experiments to probe the dynamics of the NcNTD in solution. While these experiments are not yet fully complete, they reveal interesting information regarding differences in the monomer-to-dimer transition between the WT and D17A mutant. Based on these analyses, I propose that the presence or absence of the D17-D53 handshake alters the topology of the NTD monomer subunit, which increases the heterogeneity of the possible conformers the NTD subunits can adopt. This variability contributes to the model of conformational selectivity, in which the NTD subunits populate many conformations that are in dynamic equilibrium and dimerize when a monomer subunit selects a partner with the complementary binding interface. The intermolecular salt bridges at either end of the dimer interface serve to properly align the subunits, and the plasticity they impart allows for the various combinations of subunit pairs to form dimers. This dissertation contributes to the understanding of the detailed mechanisms that govern the spidroin NTD dimerization process that is critical to spider silk formation.

Rights

All rights reserved. Copyright is held by the author.

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