Date of Award

Spring 2017

Project Type

Thesis

Program or Major

Biochemistry

Degree Name

Master of Science

First Advisor

Feixia Chu

Second Advisor

Rick Cote

Third Advisor

Estelle Hrabak

Abstract

Cross-linking coupled to Liquid Chromatography/Mass Spectrometry (CXMS) has become an invaluable technique for examining protein-protein interactions and monitoring enzymatic conformational changes. Thus far, comparative quantitation of cross-linked peptides measured by their mass spectrometry (MS) ion intensity is employed to deduce residue cross-linking propensity and spatial proximity. Using two distinct conformations of a structurally well-characterized model protein, we examined the correlation of cross-linked peptide MS signals and distances, but were not able to observe any obvious correlation. Conceivably, several physiochemical factors can affect residue reactivities thus MS signal intensity of the corresponding cross-linked peptides. For NHS ester chemical cross-linkers, the NHS ester functionality often undergo hydrolysis simultaneously to produce dead-end cross-links. Importantly, these dead-end cross-links have proved to be a reliable measurement of the cross-linked lysine residues. Therefore, we propose a novel analytical method by which these dead-end cross-links provide important contextual information about the amino acid side chains involved in cross-linking reactions. Normalization of cross-linked peptide ion intensity against the cognate dead-end cross-links yields a value (proportional cross-link intensity) which can provide a basis for comparison across different biochemical conditions or enzyme interaction states. In this work, we use this method in conjunction with isotopically labeled cross-linking reagents to examine the conformational changes on the part of HtpG (the E. coli homolog of Hsp90) and a truncation mutant of one of its client proteins, Staphylococcal Nuclease. Dramatic conformational changes are observed in the client protein as HtpG transitions through its ATPase cycle. Next, we apply label-free quantitation and the proportional cross-link intensity model to the vertebrate retina enzyme Phosphodiesterase 6 (PDE6). Through pair-wise combinations of the catalytic subunits, cyclic GMP, and the inhibitory subunits, we observe conformational changes in response to allosteric binding of regulatory subunits and molecules.

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