Ultrafast protein conformational alterations in bacteriorhodopsin and its locked analogue BR5.12
Bacteriorhodopsin, reconstituted with a sterically "locked" retinal chromophore, BR5.12, has frequently been used to elucidate elementary photoinduced processes in the native pigment bacteriorhodopsin. In this work, the vibrational response of BR5.12 to photoexcitation is investigated by means of femtosecond time-resolved mid-infrared and UV-vis spectroscopy. The electronically excited state of BR5.12 decays with a time constant of 18 ps. Neither in the UV-vis nor in the mid-IR spectral region are indications found for chromophore photoproducts, besides the full recovery of the electronic ground state. However, vibrational bands are observed at around 1660 and 1550 cm-1 in the protein amide I and amide II band regions, respectively. They are formed within a few picoseconds or even instantaneously. Thus, they appear faster than the Si decay and persist for at least 130 ps, i.e., for much longer than the Si lifetime. These findings strongly suggest that the observed bands must be assigned to protein vibrations and that they are not caused by a photoinduced temperature rise. Thus, for the first time, ultrafast protein vibrational changes are detected in BR5.12, that are not associated with isomerization. Possibly they can be related to the enhanced chemical reactivity of photoactivated BR5.12 reported in the literature. In wild-type bacteriorhodopsin, bands with very similar spectral and kinetic characteristics are observed, suggesting that they might originate from a similar mechanism which is not isomerization. A plausible mechanism is a polarization induced protein conformational change, as discussed in the literature.