Date of Award

8-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics and Biochemistry

Committee Member

Dr. William Marcotte, Committee Chair

Committee Member

Dr. James Morris

Committee Member

Dr. Michael Sehorn

Committee Member

Dr. Kerry Smith

Abstract

One of the most interesting biomaterials known to man is spider silk. These fibers, composed primarily of protein, show beneficial mechanical and biological properties that have many uses in medical and industrial fields. Spider silk proteins (spidroins) are composed of three core components that consists of a C-terminal domain (CTD), repeat domain (R), and N-terminal domain (NTD). The NTD and CTD are highly conserved and are believed to form “multimeric strands” that make up spider silk. Although it is known that the NTD undergoes a conformational change that results in stable homodimer formation at acidic pH , the way in which the NTD contributes to fiber assembly is still under investigation. .

Here we provide label-free, real-time dimerization data of the wild-type major ampullate spidroin 1 (MaSp1) NTD from Nephila clavipes using the Octet RED96 system. This data shows over 25-fold increase in the affinity of wild-type NTD monomers at pH 5.5 compared to pH 7.0. These results are in agreement with previous work from our lab and others that show an increase in homodimer stability at acidic pH. This information correlates with the conformational change seen as the wild-type NTD transitions from a neutral to acidic pH environment. We found that NTD variants D45K and E84K abolished the conformational change that occurs at pH 5.5 and decreased dimer stability compared to wild-type MaSp1 NTD. We also found showed that variant K70D and double variant D45K/K70D exist in an intermediate conformation between that seen at pH 5.5 and pH 7.0 for wild-type NTD. This new information provides additional evidence to support the idea that a conformational change is directly connected to dimerization. Our data provides additional insight into which residues are necessary for this conformational change and subsequent dimerization, which allows for a better understanding of the homodimerization process.

We also demonstrate production of MaSp2 “mini spidroins” in Saccharomyces cerevisiae. These “mini spidroins” contained a native CTD and NTD and eight repeat units and preliminary results show that glucose and raffinose preinduction media result in better protein expression using 24 hr and 72hr induction times, respectively.

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