Date of Award

2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Harry Ischiropoulos

Abstract

ABSTRACT

THE STRUCTURAL PROTEOMICS OF S-NITROSYLATION: FROM GLOBAL IDENTIFICATION TO ELUCIDATING PROTEIN FUNCTION THROUGH STRUCTURAL BIOINFORMATICS

Jennifer L. Greene

Harry Ischiropoulos, Ph.D.

S-nitrosylation is the covalent addition of nitric oxide to reduced cysteine residues on proteins. It has been well documented that not all proteins are S-nitrosylated and more specifically, not all cysteine residues within an S-nitrosylated protein are modified. Therefore, it is very important to determine how this specificity is derived. Additionally, the mechanism by which nitric oxide can modify cysteines is still unclear. Even with the discovery of functional consequences of S-nitrosylation, there are still large deficits in our understanding and validation that it is a newly identified means of nitric oxide signaling within the body. These gaps in knowledge primarily exist due to a lack of tools necessary for identifying in vivo sites of S-nitrosylation. To this end, complementary mercury-based mass spectrometric approaches were developed for the identification of endogenous S-nitrosoproteomes. This resulted in the identification of 328 SNO-cysteines coordinated to 192 proteins in the mouse liver, 97% of which corresponded to novel targets of S-nitrosylation. Bioinformatic analysis of these targets then revealed that multiple mechanisms of S-nitrosylation may occur in vivo, one of which involving S-nitrosoglutathione (GSNO). To test this hypothesis, the SNO-proteome of mice incapable of metabolizing GSNO was resolved. Quantum mechanics/molecular mechanics calculations coupled with molecular dynamics simulations proposed a novel GSNO-mediated mechanism of transnitrosation. Basic residues in the surrounding cysteine microenvironment were shown to catalyze the formation of protein S-nitrosocysteine residues. Collectively, these data suggest that the specificity of cysteines targeted for S-nitrosylation is driven by the surrounding protein microenvironment. Additionally, with only 9 structures of S-nitrosylated proteins our present understanding of the structural consequences of S-nitrosylation is limited. Using an in vivo model, attempts were made to correlate changes in enzymatic activity as a function of S-nitrosylation. Normal mode analysis revealed local motions near the site of S-nitrosylation which may alter product release. In summary, this thesis utilized a global proteomic approach to craft a more targeted investigation into the specificity and molecular mechanism of S-nitrosylation.

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