Date of Award

2019

Document Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Craig Vierra

First Committee Member

Geoff Lin-Cereghino

Second Committee Member

Jane Khudyakov

Abstract

Spider dragline silk is a biomaterial with outstanding material properties, possessing high-tensile strength and toughness. In nature, dragline silk serves a central role during spider locomotion and web construction. Today, scientists are racing to elucidate the molecular machinery governing silk extrusion, attempting to translate this knowledge into a mimicry process in the laboratory to create synthetic fibers for a wide range of different applications. During extrusion, it has been established that biochemical and mechanical forces govern spidroin folding, aggregation, and assembly. In black widow spiders, at least 7 different proteins have been identified as constituents of dragline silk fibers. These represent the major ampullate spidroins, MaSp1 and MaSp2, and several low-molecular weight cysteine-rich protein (CRP) family members that include CRP1, CRP2, and CRP4. Molecular modeling studies have predicted that CRPs contain a cystine slipknot motif. To advance scientific knowledge regarding CRP function, we expressed and purified recombinant CRP1 and CRP4 from bacteria and investigated their secondary structure using circular dichroism (CD) under different chemical and physical conditions. We demonstrate by far-UV CD spectroscopy that CRP1 and CRP4 contain similar secondary structural characteristics in solution, displaying substantial amounts of random coil conformation, followed by lower levels of beta sheet, alpha helical and beta-turn structure. Additionally, we show that native structures of CRP1 and CRP4 have high thermal stability and are resistant to conformational changes under acidic pH conditions. Taken together, the chemical and thermal stability of CRP1 and CRP4 are experimentally consistent with biochemical properties of cystine slipknot proteins.

Pages

34

Available for download on Thursday, October 31, 2019

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