OPTICAL STUDIES OF A BACTERIAL PHOTORECEPTOR PROTEIN, PHOTOACTIVE YELLOW PROTEIN, IN SINGLE-CRYSTALS

Photoactive yellow protein (PYP), isolated from Ectothiorhodospira hal ophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin II. We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms. The PYP crystal has a bright yellow color and d isplays pronounced anisotropic absorption properties. Linear dichroism measurements show that the transition moment of the PYP chromophore m akes an angle of 73 degrees (or 107 degrees) with respect to the six-f old crystallographic symmetry axis. The crystal absorbance can be blea ched reversibly as indicated by absorption changes. A bleached photost ationary state in the crystal can be established via CW laser illumina tion, and the extent of crystal bleaching is found to be clearly depen dent on excitation laser wavelength, intensity and illumination time. These results provide the information for designing time-resolved crys tallography experiments in which a minimum perturbation is applied to the PYP crystals. Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 degre es C; a slower component of 1.4 +/- 0.2 s(-1) accounts for 60% of the absorbance change and a faster component of 5.2 +/- 0.9 s(-1) for the other 40%. As a control, we found that the kinetics for the same relax ation in solution are well described by one exponential and agree quan titatively with previous studies. The two rate constants observed in t he crystal show similar temperature dependences, with activation energ ies for the slow and fast components of 11.7 +/- 1.2 and 5.5 +/- 2.3 k cal/mol, respectively. However, the amplitudes associated with the two exponents show different and opposite temperature dependence. Our res ults show that the solution kinetic model is not directly applicable t o crystals. A kinetic model consistent with the optical data is import ant to extract the underlying structural intermediates from the time-r esolved X-ray diffraction data obtained in parallel with the optical d ata described here. We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.