INVESTIGATION OF HNO-INDUCED MODIFICATIONS

IN VARIOUS SYSTEMS

by

Gizem Keceli

A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy

Baltimore, Maryland

May, 2014

© 2014 Gizem Keceli All Rights Reserved

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Abstract

Nitroxyl (HNO), a potential heart failure therapeutic, is known to post-

translationally modify cysteine residues. Among reactive nitrogen oxide species, the

modification of cysteine residues to sulfinamides [RS(O)NH2] is unique to HNO. Because

this modification can alter protein structure and function, we have examined the reactivity

of sulfinamides in several systems, including small organic molecules, peptides, and a

protein. At physiological pH and temperature, relevant reactions of sulfinamides involve

reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids

[RS(O)OH]. In addition to utilizing ESI-MS and other spectroscopic methods to

investigate sulfinamide reduction, we have applied 15N-edited 1H-NMR techniques to

sulfinamide detection and used this method to explore sulfinamide hydrolysis.

Since HNO-derived modifications may depend on local environment, we have also

investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-

containing peptides. Apart from the lack of sulfinamide formation, these studies have

revealed the presence of new products, a sulfohydroxamic acid derivative [RS(O)2NHOH]

and a thiosulfonate [RS(O)2SR], presumably produced under our experimental conditions

via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH).

Apart from its role in thiol oxidation, HNO has been reported to have nitrosative

properties, for example with tryptophan resulting in N-nitrosotryptophan formation. We

have examined the reactivity of HNO with tryptophan and small peptides containing either

tryptophan or both a tryptophan and a cysteine residue.

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HNO has been shown to enhance cardiac sarcoplasmic reticulum Ca2+ cycling

independent of the -adrenergic pathway. In a collaborative project, the effects of HNO

on the cardiac proteins, phospholamban (PLN) and sarco(endo)plasmic reticulum Ca2+-

ATPase (SERCA2a) were investigated.

Advisor: Professor John P. Toscano

Readers: Professor Mark M. Greenberg

Professor Craig A. Townsend

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Acknowledgments

First and foremost, I would like to thank my advisor, Professor John P. Toscano,

for providing me the research opportunity in his laboratory and also for all his help

throughout my studies. I sincerely appreciate and respect his strive for excellence, and also

would like to express my gratitude for his patience, tolerance, optimism, and understanding

over the years. It has always been a great pleasure to work with him.

I would also like to acknowledge Professor Mark M. Greenberg and Professor Craig

A. Townsend for serving on my thesis committee and reading my dissertation.

I am very grateful to our collaborator, Professor Nazareno Paolocci (Johns Hopkins

Medical Institutions), who has been like a co-advisor to me since I joined Toscano group.

So I would like to thank him for all his help and guidance over the years.

I thank to our collaborators Professor James E. Mahaney (Edward Via Virginia

School of Osteopathic Medicine) and the late Professor Jeffrey P. Froehlich (Johns

Hopkins Medical Institutions) on the phospholamban project, and Professor Michael P.

Yassa on the caffeine project. I really appreciate the support of Professor Naod Kebede

(Edinboro University) in synthesis and Professor Tamara L. Hendrickson (Wayne State

University) in biochemistry protocols and peptide synthesis. I would also like to recognize

Dr. Jason W. Labonte for conducting the computational studies and Daniel Borota for his

work in the caffeine project. The facility managers, Dr. I. Phillip Mortimer and Dr. Cathy

D. Moore, have been extremely helpful throughout my studies and I would like to thank

them for teaching me MS and NMR experiments, respectively.

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I appreciate the support and input of all the past and present members of the

Toscano group. In chronological order, I thank Dr. Christopher M. Pavlos (for his previous

work on phospholamban), Dr. Andrew D. Cohen (for showing me around the office), and

Dr. Yonglin Liu. I owe special thanks to my labmates with whom we have gone through

all the ups and downs of graduate school, Dr. Anthony S. Evans, Dr. Art D. Sutton, and

Dr. Daryl A. Guthrie, as well as the new members of our lab, Christopher Bianco, Tyler

Chavez, Hyunah Cho, and Saghar Nourian for all the useful conversations and their

friendship.

Also I would like to thank all my friends for their support and encouragement, and

acknowledge everyone who has helped me one way or the other over the years. Lastly,

thanks to my parents, and my brother in Turkey for always supporting me in all my

decisions.

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Table of Contents

Abstract……………………………………………………………………………………ii

Acknowlegments…………………………………………………………………………iv

Table of Contents…………………………………………………………………………vi

List of Tables………………………………………………………………………….....xii

List of Figures……………………………………………………………………………xii

List of Schemes………………………………………………………………………….xxi

Chapter 1. Introduction…………………………………………………………………...1

1.1 Nitroxyl (HNO)………………………………………………………………..1

1.2 Effects of HNO on Ca2+ Cycling……………………………………………...3

1.3 Reactivity of HNO…………………………………………………………….6

1.4 Introduction to This Work…………………………………………………...10

1.5 References…………………………………………………………………...12

Chapter 2. Reactivity of HNO-Derived Sulfinamides…………………………………..21

2.1 Introduction…………………………………………………………………..21

2.2 Results………………………………………………………………………..25

2.2.1 Formation of Peptide Sulfinamides by Reaction with HNO………25

2.2.2 Sulfinamide Reduction…………………………………………….28

2.2.3 Sulfinamide Reduction in Peptides versus a Small Organic Molecule

as a Function of Solvent Dielectric Constant…………………………….33

2.2.4 Deamidation of Asn-Containing Peptides…………………………36

2.2.5 Conversion of Sulfinamides to Sulfinic Acids……………………..36

2.2.6 Detection of Ammonia……………………………………………..39

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2.2.7 Reduction of Sulfinamide Modification in Papain………………...40

2.3 Discussion……………………………………………………………………42

2.4 Conclusions…………………………………………………………………..45

2.5 Experimental Methods……………………………………………………….45

2.6 References……………………………………………………………………52

2.7 Supporting Information………………………………………………………61

Chapter 3. NMR Detection and Study of the Hydrolysis of HNO-Derived

Sulfinamides……………………………………………………………………………..67

3.1 Introduction…………………………………………………………………..67

3.2 Results and Discussion………………………………………………………69

3.2.1 Detection of Synthetic and HNO-Derived Small Organic Molecule

Sulfinamides by 1H NMR………………………………………………..70

3.2.2 15N-Edited 1H 1D NMR to Detect 15N-Labeled Small Organic

Molecule Sulfinamides…………………………………………………..73

3.2.3 Detection of HNO-Derived Sulfinamides in Peptides……………..74

3.2.4 Detection of an HNO-Derived Sulfinamide in Papain…………….78

3.2.5 Reduction of Peptide Sulfinamides………………………………...81

3.2.6 Hydrolysis of Peptide Sulfinamides………………………………..81

3.2.7 Hydrolysis of Sulfinamide-Modified Papain and Comparison with Its

Model Peptide……………………………………………………………83

3.3 Conclusions…………………………………………………………………..87

3.4 Experimental Metho