| Abstract
| - Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-statePYP (PYPdark) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonanceRaman spectra of PYP with 225−250 nm excitations and investigate protein structural changes accompanyingthe formation of the putative signaling state denoted PYPM. The PYPM−PYPdark difference spectra showseveral features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues.The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Althoughthere are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the differencesignals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengthsand the crystal structure of PYPdark. A further experiment on the Thr50→Val mutant supports environmentalchanges in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond betweenTyr42 and the chromophore in PYPM. In addition, a reorientation of the OH group in Tyr42 is suggestedfrom the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminishin intensity upon formation of PYPM. The loss of intensities is attributable to an exposure of tryptophan inPYPM. PYP contains only one tryptophan (Trp119) that is located more than 10 Å from the active site. Thusthe observed changes are indicative of global conformational changes in protein during the transition fromPYPdark to PYPM. These results are in line with the currently proposed photocycle mechanism of PYP.
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