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Date Permissions Signed

7-15-2013

Date of Award

2013

Document Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Smirnov, Sergey L.

Second Advisor

Spiegel, P. Clint

Third Advisor

Anthony-Cahill, Spencer J.

Abstract

The protein villin is a member of the gelsolin superfamily, and regulates cytoskeleton formation in the brush border at the apical end of epithelial cells. The six villin domains (V1-V6) form which contain a Ca²+ -dependent actin-binding site associated with the V1-V3 fragment. The six villin domains are homologous to gelsolin and have ~50% sequence identity. In the absence of calcium, gelsolin adopts a compact, inactive conformation stabilized by a 12-residue C-terminal helix. This helix locks domains G2 and G6 as a "latch" in low calcium and releases at higher calcium levels. Tyrosine phosphorylation, phosphatidylinositol 4,5-bisphosphate (PIP2) and calcium concentration control villin's function to cleave, nucleate, cap and bundle F-actin. Villin bundles F-actin in low calcium and severs it in high calcium to regulate formation of microvilli. Domain six of villin (V6) is connected by a 40 residue unstructured linker to the actin-binding C-terminal headpiece domain (HP). The headpiece is required for F-actin bundling and is calcium insensitive. The construct V6-HP has been shown to bundle F-actin as a monomer in high calcium. The mechanism for how intact villin bundles is unclear, with two main hypothesis proposed. One of these states that monomeric villin bundles using one F-actin binding site on HP and another, potentially cryptic binding site on one of the core domains. The alternative hypothesis states that villin dimerizes via V2 and uses two headpieces to bundle F-actin. We show that the V6-linker construct made up of domain six of villin (V6) with the 40 residue unstructured linker (which connects to HP) does not bind F-actin in high or low calcium. We also report the preliminary structural characterization of V6 in the absence of calcium, which adopts a gelsolin-like fold and suggests a different conformation of the long a-helix than that in the presence of high calcium. These data are consistent with analogous calcium activation and helix- straightening mechanism of gelsolin, in light of the previously determined solution structure of V6 at 5mM calcium with a straight long a-helix. V6-linker and V6 were found to be monomeric in high calcium and slowly form aggregates in calcium-free environment. This data suggests that dimeric villin bundles F-actin and highlights the significance of V6 and the unstructured linker in the regulation of intact villin. In addition, our study looks at supervillin, the largest member protein, shown to increase cell motility and promote invasive activity in tumors. The N-terminus of supervillin (amino acids 1-830) interacts with the major cytoskeletal components of filamentous actin and mysoin II. We utilized circular dichroism (CD) and bioinformatics sequence analysis to demonstrate that the N-terminal part of supervillin forms an extended intrinsically disordered region (IDR). We show the N-terminus (a.a. 1-830) of human and bovine supervillin sequences to be an intrinsically disordered region, making it the largest currently known IDR to date in the villin/gelsolin family. Additionally, we propose a novel mechanism of regulation of myosin II and F-actin via the intrinsically disordered N-terminal region of supervillin. Further work will explore the structure/function and dynamics of domains 2 and 3 (SV2-3) of supervillin as well as the homologous V6-HP construct in supervillin (SV6-HP).

Type

Text

DOI

https://doi.org/10.25710/50xt-z035

Publisher

Western Washington University

OCLC Number

856583714

Subject – LCSH

Microfilament proteins--Physiological effect; Actin; Brush border membrane; Carrier proteins; Binding sites (Biochemistry)

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document 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.

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