Effects of Modification of Protein Nanospace Structure and Change of Temperature on the Femtosecond to Picosecond Fluorescence Dynamics of Photoactive Yellow Protein

The effect of the protein environment surrounding a chromophore [protein nanospace (PNS)] on the photoinduced ultrafast reaction of the chromophore (deprotonated p-coumaric acid covalently bonded to the protein via a thioester linkage) has been investigated by comparing the femtosecond to picosecond fluorescence dynamics of wild-type (w-t) photoactive yellow protein (PYP) with those of the chromophore in aqueous solution as well as in various PNSs modified by site-directed mutagenesis. The fluorescence decay dynamics of the chromophore are considerably accelerated in PNSs compared with those in solution. The ultrafast nonexponential fluorescence decay dynamics of the w-t PYP in the 100 fs to 10 ps time regime, which are probably due to the formation of a twisted state by the flipping of the thioester linkage of the chromophore, have been slowed in the mutants, which seem to have a looser PNS structure because of the weakening of the chromophore-amino acid residue hydrogen-bonding interaction and the partial destruction of the H-bonding network surrounding the chromophore in PNS by mutation. Specifically, the more restricted structure of the PNS in w-t PYP seems to be favorable for twisting by the flipping mechanism. In addition, we have examined temperature effects on the fluorescence dynamics of the w-t PYP and several mutants to elucidate the mechanisms underlying the highly nonexponential decay of the w-t PYP as well as the remarkable influences on the fluorescence dynamics of modifications in the PNS structure by site-directed mutations. We have revealed by these investigations that the fastest decay in the femtosecond to 1 ps regime in w-t PYP is temperature-independent and that the twisting reaction responsible for it could be of a coherent or barrierless origin, whereas thermal activation is necessary for the reaction in the few picosecond to 10 ps regime. For the mutants, however, no activationless process could be observed. Therefore, rapid relaxation into traps seems to occur, competing with the barrierless reaction in the case of the w-t PYP and becoming overwhelming in the mutants. After such trappings, slow reactions may take place by thermal activation.