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Date Permissions Signed

7-21-2022

Date of Award

Summer 2022

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Smirnov, Sergey L.

Second Advisor

Antos, John M.

Third Advisor

Young, Jeff C. (Jeffery C.)

Abstract

A reported 33% of eukaryotic proteins are predicted to contain intrinsically disordered regions (IDRs) over 30 residues in length. IDRs are regions of protein which natively exist in an unfolded conformation. Due to their highly dynamic nature, many common methods of inquiry such as crystallography and NMR can be thwarted. As a result, valuable analysis such as probing function, dynamics and binding interfaces are unable to be performed. To minimize these problems, researchers typically study shorter IDRs. We probed fragments of plant protein villin 4 (VLN4) for covalent modifications to explore its regulation and degradation. Disordered regions of proteins have greater solvent accessibility compared to their folded counterparts. This results in various post translational modifications, a common method of protein regulation. The protein VLN4 is vital to the development of roots and understanding its regulation could greatly benefit agriculture. Thus, VLN4 was examined for degradation, ubiquitination, and phosphorylation.

Complimentarily, overcoming challenges of IDR research can be done by targeting the IDR with covalent modification via sortase-mediated ligation (SML). In NMR, large IDRs create substantial spectral overlap in the 1H dimension for backbone amide protons. By introducing isotopically labeled residues with SML, this overlap can be greatly reduced and allow for characterization of longer IDRs. Through the use of authentic IDRs with extreme pentapeptide residue properties on N- and C- terminal flanking regions of LPXTG motifs, ligation efficiencies were characterized, followed by identification and optimization of problematic sortase ligation sequences.

Type

Text

Keywords

Intrinsically, Disordered, Proteins

Publisher

Western Washington University

OCLC Number

1338692406

Subject – LCSH

Plant proteins--Analysis; Proteins--Chemical modification; Prokaryotes--Analysis; Solvation

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 document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

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