  Bacteriorhodopsin (bR) is a light-driven proton pump. We use time-resolved
crystallography at an X-ray free-electron laser to follow the structural
changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds.
Quantum chemistry and ultrafast spectroscopy allow identifying a sequential
two-photon absorption process, leading to excitation of a tryptophan residue
flanking the retinal chromophore, as a first manifestation of multi-photon
effects. We resolve distinct stages in the structural dynamics of the all-trans
retinal in photoexcited bR to a highly twisted 13-cis conformation. Other
active site sub-picosecond rearrangements include correlated vibrational
motions of the electronically excited retinal chromophore, the surrounding
amino acids and water molecules as well as their hydrogen bonding network.
These results show that this extended photo-active network forms an
electronically and vibrationally coupled system in bR, and most likely in all
retinal proteins.
