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Date of Award

Spring 2024

Document Type

Masters Thesis

Department or Program Affiliation


Degree Name

Master of Science (MS)



First Advisor

Smirnov, Serge L.

Second Advisor

Antos, John M.

Third Advisor

Amacher, Jeanine


The postulate that structure informs function has driven protein science for many years. This paradigm has since shifted, and examples of intrinsically disordered proteins (IDPs) or proteins containing novel intrinsically disordered regions (IDRs) demonstrate that these dynamic polypeptides perform many critical functions. Here, we have explored the F-actin bundling capabilities of A. thaliana villin-4, which is a protein that is critical for nutrient and moisture uptake from the soil via root hairs. Despite the experimental challenges of working with IDR systems, we were able to determine that A. thaliana villin-4’s C-terminus contains a novel F-actin bundling segment. When combined with the remainder of the C-terminus, the complete polypeptide sequence acts as a self-regulating segment which bundles F-actin via electrostatic and hydrophobic interactions, forces which contributes to varying degrees depending on ionic strength and temperature. By understanding villin-4’s C-terminus, we speculate that the root systems of plants can be improved to enhance crop yields, either by optimization in a controlled environment (greenhouses) or by direct intervention via protein engineering.

In addition to studies of villin 4, this thesis has also explored the continued development of sortase mediated ligation (SML) as a tool for protein engineering. Recent optimizations of this technique include, but are not limited to, mutating the enzyme to the now famous Heptamutant Sortase A (SrtA7M) and altering sequence context around the LPXTG recognition motif using spacers. However, so far there have been no in-depth studies on the effect of chemical composition upstream of LPXTG with the aim of identifying highly specific/efficient sortase substrates. Here, we show that a largely polar and charged five amino acid context with a hydrophobic residue at its core, acts as a superior SrtA7M substrate when directly N-terminal to the LPXTG motif. This allows for the efficient production of SML ligation products derived from a biomedically relevant protein target (DARPin). This finding has practical implications for the sortase protein engineering field as it allows for the efficient and specific installation of useful modifications with downstream applications in fundamental biochemistry and biomedical research.




Villin 4, Heptamutant Sortase A, Protein Engineering, SML, DARPin, Actin Bundling, Co-sedimentation Assay, NMR, IDRs


Western Washington University

OCLC Number


Subject – LCSH

Plant proteins; Enzymes--Analysis; Protein engineering; Microfilament proteins; Actin




masters theses




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