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 containing a thiol ester-linked p-coumarate anion chromophore. Photoexcitation of PYP triggers a photocycle which involves at least two intermediates: 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 blue-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 pK of 2.8 and a steepness of 1.35 and report that the formation of pB(dark) is also induced by increased pressure (midpoint similar to 1250 atm 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 the data we calculated the UV/Vis absorbance spectral and kinetic properties of pB(dark) and pB, together with their pH dependencies between pH 5 and 2. Similarities between pB and pB(dark) were found with respect to their absorbance spectra in both the UV and visible region and with respect to the effect of pH on their stability. It is proposed that an increase in acidity and/or pressure leads to the steady state partial unfolding of PYP, while photoexcitiation leads to an analogous but transient unfolding process. (C) 1997 Elsevier Science B.V.