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Date Permissions Signed

8-1-2022

Date of Award

Summer 2022

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry Department

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Smirnov, Sergey L.

Second Advisor

Antos, John M.

Third Advisor

Anthony-Cahill, Spencer J.

Abstract

About 43% of eukaryotic proteins contain intrinsically disordered regions (IDRs) of 40 residues or longer. These proteins modulate key cellular functions such as transcription, translation, and signal transduction. Despite their vital roles, little is known about the properties of large disordered proteins due to the difficulties encountered studying them. Solution nuclear magnetic resonance (NMR) spectroscopy is a unique tool allowing for their analysis but traditionally requires uniform protein labeling with 15N and 13C isotopesresulting in convoluted spectra which obscure any meaningful information on their structures and interactions. To remedy this issue, we aim at developing a procedure to segmentally label large IDR-containing proteins by synthesizing them as separate fragments before ligating them together using sortase enzymes. This method would allow for the recording of 15N/13C-HSQC experiments of smaller IDR/IDP segments at a time, avoiding spectral overlap while retaining the proteins' native biochemical environment. We focus our work on cytoskeletal regulators, dematin (Homo sapiens) and villin 4 (Arabidopsis thaliana) each comprising a large IDR. Both dematin and villin 4 bind and control the bundling of filamentous actin (F-actin) but their mechanism of action remains unclear. We plan to use segmental labeling (SL) to gain insight into their respective properties which would help advance both the medical (dematin) and agricultural (villin 4) fields.

Type

Text

Keywords

IDR, dematin, villin, urea, SML, segmental labelling, sortase, NMR, purification, SLIDR

Publisher

Western Washington University

OCLC Number

1340125020

Subject – LCSH

Cytoskeletal proteins; Prokaryotes; Eukaryotic cells; Nuclear magnetic resonance spectroscopy

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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