Protein S-nitrosylation, or the attachment of an NO group to the sulfur of a cysteine side chain (SNO) within a protein, is now recognized as an important mechanism for the post-translational regulation of protein function. S-nitrosylation is known to regulate the function of every major class of protein and to be involved in a variety of human pathophysiologies. The field of S-nitrosylation biology is expanding rapidly, but currently little work is being done to understand the consequences of this post- translational modification from a structural perspective. We propose here to first examine the chemical and structural """"""""rules"""""""" that dictate the specificity of protein S-nitrosylation. To accomplish this we will use computational methods to search for similarities in the charge distribution, hydrophobicity, secondary structure, etc. of the immediate environment surrounding cysteines that are known sites of S-nitrosyaltion. We will use the human thioredoxin protein as a model system to validate motifs we uncover computationally, making specific mutations surrounding the S-nitrosylated cysteines of thioredoxin and monitoring the reactivity and stablilty toward S-nitrosylation relative to the wild-type protein. We will also study the structural and functional consequences of protein S-nitrosylation in several specific protein systems. We have previously demonstrated using X-ray crystallography that S- nitrosylation of myoglobin induces a conformational change in that protein. We will now examine the functional consequences of this conformational change on oxygen binding to myoglobin using spectroscopic methods and computational simulation of myoglobin dynamics. A recently discovered S- nitrosylation-induced cell death cascade involves the S-nitrosylation-dependent association of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the ubiquitin ligase Siah. We will observe via X-ray crystallography the allosteric changes in GAPDH induced by S-nitrosylation that modulate protein-protein interactions with Siah. We will also study binding of deprenyl and related compounds to GAPDH, which block the S-nitrosylation-induced cell death cascade. Nitric oxide synthase (NOS) is the enzyme responsible for production of nitric oxide within cells, without which no protein S-nitrosylation could take place. It has been shown that NOS can undergo S-nitrosylation during normal catalytic turnover, and we have previously demonstrated via X-ray crystallography that the tetrahydrobiopterin cofactor of inducible NOS (iNOS) can undergo a novel N-nitrosylation. In this work we will examine the structural and functional effects of both S- and N-nitrosylation on the three NOS isoforms. The experiments proposed here will directly contribute to the understanding of S-nitrosylation specificity as well as its structural and functional effects on proteins. This information will aid the design of therapeautic treatments to disorders involving aberrant protein S-nitrosylation. Public Health Relevance: Relevance Protein S-nitrosylation is now recognized as an important mechanism for regulating protein function, and abberant S-nitrosylation may play a role in human disease. The experiments proposed here will directly contribute to the understanding of S-nitrosylation specificity as well as its structural and functional effects on proteins. This information will aid the design of therapeautic treatments to disorders involving aberrant protein S-nitrosylation.
Protein S-nitrosylation is now recognized as an important mechanism for regulating protein function, and abberant S-nitrosylation may play a role in human disease. The experiments proposed here will directly contribute to the understanding of S-nitrosylation specificity as well as its structural and functional effects on proteins. This information will aid the design of therapeautic treatments to disorders involving aberrant protein S-nitrosylation.
| Alicea, Ismael; Marvin, Jonathan S; Miklos, Aleksandr E et al. (2011) Structure of the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors. J Mol Biol 414:356-69 |
