The vast majority of theses in this collection are open access and freely available. There are a small number of theses that have access restricted to the WWU campus. For off-campus access to a thesis labeled "Campus Only Access," please log in here with your WWU universal ID, or talk to your librarian about requesting the restricted thesis through interlibrary loan.
Date Permissions Signed
Date of Award
Master of Science (MS)
Smirnov, Serge L.
Anthony-Cahill, Spencer J.
Spiegel, P. Clint
The villin family of proteins are filamentous actin (F-actin) regulators, which play key roles in cytoskeleton organization regulation in eukaryotes. Recently discovered villin 4, from A. thaliana has been shown to bundle F-actin in root hairs. However, previous work found that mutations to villin 4 result in shorter and sparser root hairs which lead to a weaker capacity for water absorption by A. thaliana. It is previously known that many members of the vertebrate villin protein family contain a highly conserved F-actin binding site within the C-terminal headpiece domain. In order to investigate the headpiece of villin 4, we predicted where the folded domain began based on sequence homology. Interestingly, in plant villins, a long linker (> 100 residues) region exists between the folded core domain and the C-terminal headpiece. While it is known that vertebrate villin contains a 40-residue linker, it shows no homology to that of the plant linkers and, therefore, the linker-headpiece boundary in villin 4 was unclear. To define this boundary, we designed two constructs: the first, atVHP76, we believed would contain some residues of the linker as well as the headpiece; the second construct, atVHP60, was predicted to contain just the folded headpiece domain. Furthermore, we proposed to investigate whether this portion of the linker alters the F-actin binding affinity of this domain. Presented here is the structural and functional study of the villin 4 headpiece domain (V4HP63). Our solution NMR analysis demonstrates that V4HP63 possesses a canonical villin headpiece fold, with the exception of a merged variable-loop (V-loop) and a newly discovered high-mobility loop (HM-loop). Through an actin pull-down assay, we showed that both atVHP76 and atVHP60 binds specifically and with high affinity to F-actin (Kd of 3.0 ± 0.7 µM and of 2.0 ± 1.0 µM, respectively), indicating the N-terminal portion of the linker does not alter the affinity. Comparison of the electrostatic surface potential maps of V4HP63 and high-affinity vertebrate F-actin binding headpiece domains, we propose a modification to the currently accepted rules for predicting high-affinity binding of villin headpieces. This includes a reduction of the previously described residues in the “charged crown” to K65, R37, and the C-terminal carboxylate. This work demonstrates that the plant villin 4 headpiece is evolutionarily related to the vertebrate homologs.
Western Washington University
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.
Miears, Heather, "Structural and Functional Characterization of the F-actin Binding Headpiece Domain of Plant Villin 4" (2017). WWU Graduate School Collection. 616.