Light induces destabilization of photoactive yellow protein

To understand the effect of visible light on the stability of photoactive y ellow protein (PYP), urea denaturation experiments were performed with PYP in the dark and with PYPM under continuous illumination. The urea concentra tions at the midpoint of denaturation were 5.26 +/- 0.29 and 3.77 +/- 0.19 M for PYP and PYPM, respectively, in 100 mM acetate buffer, and 5.26 +/- 0. 24 and 4.11 +/- 0.12 M for PYP and PYPM, respectively, in 100 mM citrate bu ffer. The free energy change upon denaturation (DeltaG(D)(H2O)), obtained f rom the denaturation curve, was 11.0 +/- 0.4 and 7.6 +/- 0.2 kcal/mol for P YP and PYPM, respectively, in acetate buffer, and 11.5 +/- 0.3 and 7.8 +/- 0.1 kcal/mol for PYP and PYPM, respectively, in citrate buffer. Even though the DeltaG(D)(H2O) value for PYPM is almost identical in the two buffer sy stems, the urea concentration at the midpoint of denaturation is lower in a cetate buffer than in citrate buffer. Although their CD spectra indicate th at the protein conformations of the denatured states of PYP and PYPM are in distinguishable, the configurations of the chromophores in their denatured structures are not necessarily identical. Both denatured states are interco nvertible through PYP and PYPM. Therefore, the free energy difference betwe en PYP and PYPM is 3.4-3.7 kcal/mol for the protein moiety, plus the additi onal contribution from the difference in configuration of the chromophore.