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Date Permissions Signed

10-13-2021

Date of Award

Fall 2021

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

Amacher, Jeanine

Abstract

It has long been accepted that a protein’s fold informs its function, but for the vital proteins that don’t adopt one specified fold, this is uninformative. Intrinsically disordered proteins have non-folded regions (IDRs) in their native functional state, which make up 30% of eukaryotic proteins, perform vital cellular processes and contribute to a multitude of disease states. Yet, IDR’s function, interactions and dynamics remain unknown due to their unstable, dynamic, lengthy and solvent-exposed nature. This, along with their tendency for insolubility and aggregation provide formidable challenges to not only their analysis, but their purification, requiring lengthy optimizations. NMR is valuable for IDR analysis, but limited to shorter sequences, due to severe spectral overlap characteristic of large IDRs. Segmental labeling of IDR containing proteins with NMR active isotopes reduces spectral overlap while maintaining the protein’s structural context. Segmental labeling was achieved with S. aureus sortase A via sortase-mediated ligations (SML), stitching N- and C- termini of proteins together and providing independent labeling of each protein half. This method was applied to large IDR-containing proteins; A. thaliana villin isoform 4 (V4) and H. sapiens dematin isoform 2. These understudied cytoskeleton regulators are involved in a multitude of essential cellular functions, whose analysis could have valuable implications in agriculture (V4) and human health (dematin). Segmentally labeled V4 samples allowed assignment of V4’s C-terminal IDR segment and probing of its temperature sensitivity with straightforward and expedient NMR experiments. Demonstrating this method’s compatibility with large IDRs short lifespans. SML generated isotopically segmentally labeled dematin, further proved the method’s value for IDR analysis. Segmentally labeled dematin would allow full length dematin NMR spectra to be recorded with reduced spectral overlap. This provides a means to characterize the non-covalent interface between the IDR and its phosphorylated C-terminal folded domain that halts its F-actin bundling capabilities, which represents a relevant regulatory mechanism for human health. This work demonstrates the versatility and adaptability of SML to generate segmentally labeled IDR containing proteins and its benefits for IDR analysis. This may make IDR analysis more achievable, providing valuable insights across disciplines from human health to agriculture due to IDRs essential roles within eukaryotes.

Type

Text

Keywords

Dematin, Villin, Sortase, IDR, intrinsically disordered proteins, intrinsically disordered regions, segmental labeling

Publisher

Western Washington University

OCLC Number

1277514139

Subject – LCSH

Proteins--Structure; Proteins--Analysis; Nuclear magnetic resonance spectroscopy; Proteins--Conformation; Protein engineering

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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