Inhomogeneity observed in the photocycle of photoactive yellow protein

In order to obtain insight into the hydrogen-bonding network surrounding the chromophore in biologically photo-functional proteins, photoactive yellow protein (PYP) has been modified by a combination of a site-directed mutagenesis and a substitution of the chromophore with an analogue. Fluorescence excitation and emission spectroscopies have been performed to evaluate the inhomogeneous nature of the spectra originated from the variation of conformational substates of the modified proteins. Noticeable inhomogeneity including contributions from two distinct substates has been observed for a PYP hybrid with the E46Q mutation and a substitution of the p-coumaric acid chromophore with caffeic acid. Transient absorption spectroscopy has been also conducted to clarify whether inhomogeneity affects the recovering kinetics or not. For the PYP hybrid, a remarkable difference of the recovery time from the signaling state has been observed, depending on the wavelength of the excitation light triggering the photocycle. Although this observation is preliminary, it indicates that kinetically inhomogeneous species likely exist in the PYP hybrid.