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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
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Miears, Heather, "Structural and Functional Characterization of the F-actin Binding Headpiece Domain of Plant Villin 4" (2017). WWU Masters Thesis Collection. 616.