Dissecting the conformational equilibrium of Src kinase using single-molecule FRET

Abstract

Kinases are responsible for the posttranslational phosphorylation of amino acid residues. Src non-receptor tyrosine kinases belong to the human kinase family. Src and Src family kinases were intensively investigated due to their malignant transformation and oncogenesis characters. Published crystal structures and biochemical studies indicate that Src has open and close conformation. And phosphorylation sites Y419 and Y530 are essential regulation sites responsible for the conformational change and kinase activity of Src. However, a quantitative analysis of Src regulation and conformation equilibrium has still not been conducted.\r Here we set about to provide detailed kinetic analysis and insight into conformation equilibrium for Src. As a starting point, I did ATP-NADH coupled enzymatic assays and Western blots on Src, Src catalytic domain, and phosphorylation sites mutants to provide detailed kinetic schemes for the regulation of Src. The results give quantitative information about the effect of phosphorylation sites on Src activity. Our data indicate that Src activity is not simply regulated by “on” and “off” switches corresponding to the open and close conformation. We further developed single-molecule FRET experiments to dissecting the conformational opening/closing equilibrium of Src. The FRET data suggest that Src may have an additional structure between open and close. Coupling the observed kinetic data with conformational information gives a comprehensive understanding of Src regulation.

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