Influence of the protein environment on spectroscopy and ultrafast dynamics of retinal in bacteriorhodopsin

The environment is important for the exact course of a chemical reaction. As an example of the strong influence of the environment on the reaction, this work studies the isomerization reaction of the chromophore retinal in the binding pocket of the protein bacteriorhodopsin. Three selected distance scales with reference to the chromophore are addressed: The distant protein surface at a length scale of 3-5 nm, the fuzzy environment of the binding pocket (1-2 nm) and the immediate neighborhood represented by the amino acid tryptophan Trp86 (5 Å). First, static absorption and fluorescence spectroscopies are applied to three dimensionally crystallized bacteriorhodopsin. An especially adapted set-up was developed for each task. The analysis of data is carried out in comparison with the measured spectra of the native protein membrane in solution. While the absorption spectrum shows a broadening on the high-energy side, the fluorescence spectrum deviates in the general shape of that measured in solution. The synoptic analysis of all spectra reveals the existence of three spectroscopically distinct species in the crystal: BR490 (absorption maximum at 490 nm) with a relative contribution of 28%, BR570 (native spectrum of solution, 68%), and BR610 ("blue membrane", 4%). BR490 appears particularly in the additional fluorescence band at 550 nm and is assigned to the dehydrated species of bacteriorhodopsin. The presence of that particular species indicates the suppression of rehydration in a partially dehydrated crystal and shows that the three dimensional arrangement hinders water diffusion. A simple model, close domains are assumed to form, eventually suppressing free water diffusion in a crystal. The lack of water on the protein surface modifies the protonation states of the amino acids in the binding pocket and therefore the optical properties of the retinal. The chromophore actually reacts on the alteration of its distant environment upon incorporation in the protein crystal. The second distance scale - the fuzzy environment - is addressed by a time-integrated fluorescence spectroscopy on native bacteriorhodopsin as function of the excitation wavelength (430 nm to 650 nm). The optimized experimental set-up guaranteed that the fluorescence of only the excited state I460 contributed. The measured spectra show maxima at 740 nm, and exhibit a Stokes shift of 5000 cm-1, independent of the excitation wavelength. The similarity of the fluorescence excitation spectrum with the spectrum of absorbed photons excludes energy-dependent loss channels. However, the spectra show a growing asymmetric broadening on the high-energy side with decreasing excitation wavelength. The additional fluorescence originates from vibrational hot states, also supported by the decomposition by Gaussian functions. A comparison with the fluorescence spectrum of blue membranes shows that the low-energy part also arises from vibrational hot states. Consequently, the first fast intramolecula