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Author ORCID Identifier

https://orcid.org/0000-0002-0696-0841

Document Type

Campus-Only Access for One (1) Year

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2020

Month Degree Awarded

February

First Advisor

Richard W. Vachet

Subject Categories

Analytical Chemistry | Biochemistry | Other Biochemistry, Biophysics, and Structural Biology

Abstract

This dissertation focuses on applying covalent labeling (CL) and mass spectrometry (MS) for characterizing protein-ligand complexes. Understanding protein-ligand interactions has both fundamental and applied significance. Covalent labeling is a protein surface modification technique that selectively modifies solvent-exposed amino acid side chains of proteins. A covalent bond is formed between the functional groups of labeling reagent and protein’s side chain. One of the key factors that affects CL reactivity is a side chain’s solvent accessibility. Ligand binding protects residues on the protein surface from being labeled, and residues involved in ligand binding can be indicated via decreases in labeling extents.

The main goal of this study is to develop strategies that apply CL-MS to characterize protein-ligand complexes. Diethyl pyrocarbonate (DEPC) is the labeling reagent we focused on. First, we developed a strategy that can identify ligand binding site as well as determine the ligand binding affinity to the protein. We characterized the complexes between β-2 microglobulin (β2m) and three amyloid inhibiting molecules under Cu(II)-induced amyloid forming conditions. The rest of the dissertation focused on comparing the information from two complementary MS-based methods, hydrogen deuterium exchange (HDX)-MS and CL-MS. Using three model protein-ligand systems, we demonstrate that the two labeling techniques can provide synergistic structural information about protein-ligand binding when reagents like DEPC are used for CL because of the differences in the intrinsic reaction rates of DEPC-based CL and HDX.

This dissertation highlights the power of CL-MS for characterizing protein-ligand complexes. The understanding of how three amyloid inhibiting molecules bind to Cu(II)-β2M could facilitate future library screening for new drug candidates. Our work also indicates CL-MS is capable of characterizing protein-ligand complexes that are difficult to study by other methods such as X-ray crystallography and nuclear magnetic resonance spectroscopy.

DOI

https://doi.org/10.7275/fdz4-4x24

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