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|Title:||The consequences of tyrosine modification in signalling proteins|
|Authors:||Amaning, Kwame S.|
|Presented at:||University of Leicester|
|Abstract:||Nitric oxide is a ubiquitous signalling molecule that plays a key role in cell physiology. Nitric oxide can react with a number of agents in vivo to form reactive species capable of modifying a range of biomolecules. The formation of nitrotyrosine in cells and tissues exposed to reactive nitrogen species is well documented, however, the mechanisms through which this modification affects cell function continue to be elucidated. SH2 domains are the major endogenous receptors that mediate protein- protein interactions between phosphotyrosine containing proteins and their intracellular binding partners. The interaction between a nitropeptide and the SH2 domain of the Src kinase Fyn has been investigated. Using nuclear magnetic resonance and a fluorescence resonance energy transfer based assay the dissociation constants of the high affinity phosphopeptide was determined as 40 nM, whilst the affinity of the nitropeptide and the non-modified peptide for the protein was markedly weaker (0.7 mM and 1.4 mM respectively). The chemical shift changes observed on binding of the nitropeptide, as visualised using heteronuclear single quauntum correlation spectroscopy, were similar to those generated on the binding of the phosphopeptide to 15N labelled SH2 domain. Using computer modelling, the weak binding of the nitropeptide was rationalised on the basis of a lack of key interactions between the nitrotyrosine moiety and residues that form the phosphotyrosine binding pocket of the SH2 domain. The affinity of the peptides for Fyn SH2 domains was also demonstrated in the immunoprecipitatated Fyn derived from Jurkat cells. There is evidence to suggest that in vivo protein-bound nitrotyrosine is reduced to aminotyrosine. In addition, therefore, the aminotyrosine containing peptide was investigated as being a possible substrate for Src kinase, with provisional evidence suggesting that the aminopeptide might undergo phosphorylation by Src.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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