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Date Permissions Signed

5-15-2012

Date of Award

2012

Document Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Smirnov, Serge L.

Second Advisor

Spiegel, P. Clint

Third Advisor

Anthony-Cahill, Spencer J.

Abstract

Villin is a modular, epithelial, actin binding protein responsible for the regulation of microvilli in the brush border region of the small intestine and kidney. Villin is regulated by tyrosine phosphorylation, Phosphatidylinositol 4,5-bisphosphate and calcium concentration. These signal molecules control villin's multifunctionality and allow it to switch among its several opposing functional roles. Villin can cleave, nucleate, cap and bundle F-actin. This is achieved through villin's six gelsolin-like domains which are connected by a 40 residue linker sequence to a novel C-terminal headpiece domain (HP). It is known that the HP forms a calcium insensitive F-actin binding site that is necessary for F-actin bundling. It is unclear how villin accomplishes F-actin bundling with several proposed mechanisms under debate. In this study, we determined the isolated sixth domain (D6) of villin's solution structure via NMR spectroscopy, its ability to bind calcium, its ability to oligomerize and its ability to bind F-actin. When referenced to the D6-HP fragment, the smallest known F-actin bundling sequence of villin, these data give insight into the mechanism of how D6-HP accomplishes F-actin bundling. Our study shows that D6 is monomeric and adopts a gelsolin like fold containing a five strand β-sheet sandwiched between a long α-helix and two smaller α-helices. Our 15N-HSQC data shows that D6 binds calcium and undergoes a conformational change when calcium is removed. Our sedimentation assay shows D6 possesses no capacity to bind F-actin. These data strongly point to a dominant role of the intrinsically disordered linker sequence in D6-HP's ability to bundle F-actin. Furthermore, in the context of villin as a whole, the linker sequence may play an unknown role in villin's regulation of actin structure.

Type

Text

Publisher

Western Washington University

OCLC Number

794777394

Digital Format

application/pdf

Genre/Form

Academic theses

Language

English

Rights

Copying of this thesis in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this thesis for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.

Included in

Chemistry Commons

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