Tyrosine phosphorylation-induced changes in absorption and UV resonance Raman spectra of Src-peptides

To elucidate possible conformational changes of proteins induced by tyrosine phosphorylation, the phosphorylation-induced changes in the absorption and UV resonance Raman (UVRR) spectra of the two kinds of 13-residue peptides in pp60c-src protein containing either Tyr416 (autophosphorylation site) or Tyr527 (C-terminal phosphorylation site) were investigated and compared with the changes for free L-tyrosine. Phosphorylation caused a blue shift and a marked decrease in the 275 nm absorption band (Lb transition) for both peptides and L-tyrosine. In the UVRR spectra, although wavenumber downshifts of several modes were observed upon phosphorylation, the most pronounced-change was the distinct intensity reduction of the Y1 band around 850 cm-1 and appearance of the Y13 band around 760 cm-1. These phosphorylation-induced changes were totally restored upon dephosphorylation with alkaline phosphatase. A Raman band characteristic of the Tyr527-peptide was also detected. A band at 1461 cm-1, which appeared regardless of the phosphorylation state, was assigned to amide IIp from comparison with the spectra of other peptides containing proline residues. The effects of H/D exchange on the UVRR spectra were also examined. This is the first application of UVRR spectroscopy to the detection of possible structural changes of the tyrosine residue on phosphorylation. © 1998 John Wiley & Sons, Ltd.