COMPARISON OF ACID DENATURATION AND LIGHT ACTIVATION IN THE EUBACTERIAL BLUE-LIGHT RECEPTOR PHOTOACTIVE YELLOW PROTEIN

Photoactive yellow protein (PYP) is a novel type of photoreceptor cont aining a thiol ester-linked p-coumarate anion chromophore. Photoexcita tion of PYP triggers a photocycle which involves at least two intermed iates: an early red-shifted state and a long-lived blue-shifted state (pB). At pH values below 3 PYP is reversibly converted into a stable b lue-shifted state (pB(dark)). Here we quantify the transition from pG to pB(dark) at reduced pH as a two-state transition with an apparent p K of 2.8 and a steepness of 1.35 and report that the formation of pB(d ark) is also induced by increased pressure (midpoint similar to 1250 a tm at pH 2.7). The last step in the photocycle of PYP, from pB back to pG, is strongly decelerated by acidification. By global analysis of t he data we calculated the UV/Vis absorbance spectral and kinetic prope rties of pB(dark) and pB, together with their pH dependencies between pH 5 and 2. Similarities between pB and pB(dark) were found with respe ct to their absorbance spectra in both the UV and visible region and w ith respect to the effect of pH on their stability. It is proposed tha t an increase in acidity and/or pressure leads to the steady state par tial unfolding of PYP, while photoexcitiation leads to an analogous bu t transient unfolding process. (C) 1997 Elsevier Science B.V.