Ultrafast Protein Dynamics of Bacteriorhodopsin Probed by Photon Echo and Transient Absorption Spectroscopy

Abstract

Bacteriorhodopsin (bR) is an efficient light-driven proton pump which shows a trans-cis isomerization reaction of its retinal chromophore after light absorption. BR exhibits a large reorganization energy λ of 2520 cm-1 on optical excitation. In this paper, we have studied the nature, origin, and dynamical aspects of this extensive reorganization. We report the results of a femtosecond three-pulse echo peak shift (3PEPS), transient grating (TG) and transient absorption (TA) study, complemented with those of steady-state absorption and fluorescence spectroscopy in wild-type bR and the D85S mutant in its blue and purple, halide-pumping forms. We have simulated the results in the context of the multimode Brownian oscillator (MBO) formalism. A simple model that incorporates retinal's known intramolecular vibrations, which represent 1094 cm-1 or reorganization energy, and a single Gaussian protein relaxation with a decay of 50 fs representing 1430 cm-1 of reorganization energy, yielded satisfactory results for all linear and nonlinear experimental results on wild-type bR. For the D85S mutant in its blue form, the same model could be applied with a Gaussian relaxation of 1050 cm-1 amplitude. It is concluded that the protein environment of the retinal chromophore only exhibits an inertial response, and does not show any diffusive-type motions on a sub-ps to ps time scale, which is probably a consequence of the covalently constrained, polymeric nature of the protein. Our results are in close agreement with earlier molecular dynamics simulations on bR (Xu, D.; Martin, C. H.; Schulten, K. Biophys. J. 1996, 70, 453−460), which indicated that after retinal excitation, which is accompanied by a significant charge relocation along the polyene backbone, the protein exhibits an extensive dielectric relaxation on a 100 fs time scale representing an energy change of ∼1700 cm-1. We conclude that on the sub-ps to ps timescale, the protein's major influence is electrostatic via a large number of small-amplitude motions of charges and dipoles. Major structural rearrangements of the protein do not occur on the timescale of isomerization. Polarized transient absorption measurements on bR and the D85S mutant indicated a time-independent anisotropy of the stimulated emission of 0.35, indicating that in the excited state, no change of the direction of the transition dipole moment of retinal takes place during the excited-state lifetime.

Femtosecond Stimulated Raman Study of Excited-State Evolution in Bacteriorhodopsin

“…21 , 44 -46 However, because the IVR receiving modes are not coupled to the electronic transition (i.e., Δ = 0), this process will have a negligible effect on the stimulated emission spectrum. Significant distortion of the molecular symmetry can occur by the induced torsional motion or by pyramidalization at C 14 , as has been predicted by theory. 21 As described above, a significant twist of the retinal polyene backbone in the excited state may quench the RINE signal by moving the molecule to the photochemically active region that is vertically displaced from a highly anharmonic portion of the ground-state surface, thereby causing rapid decay of the ground-state coherence following nonlinear emission.…”

“…This evolution is driven primarily by the rapid relaxation along the highly displaced degrees of freedom such as the ethylenic stretch at 1528 cm −1 (22-fs period), the C-C stretches at 1200 cm −1 (28-fs period), and the methyl rock at 1006 cm −1 (33-fs period) to a distribution of geometries centered at the excited-state equilibrium structure (step 2). 16 This ultrafast intramolecular relaxation occurs at the same time as the inertial dielectric response of the protein, 14 and together, these processes produce the <30-fs growth of the red-shifted stimulated emission band observed by us and others. 47 The multidimensional nature of this initial relaxation, in combination with rapid excited-state IVR, prevents the observation of high-frequency coherent oscillations in the excited state.…”

Resonant optical rectification in bacteriorhodopsin

“…Molecular dynamics calculations (14) and recent photon echo experiments (16) indicate that optical excitation of the retinal is followed by an intense dielectric response of the protein matrix in the 100-fs range. Our experiments show that the initial electronic polarization precedes this response, which suggests that the polarization induces the large electrostatic protein relaxation (16). Studies on bR samples reconstituted with nonisomerizable retinal (15) showed that retinal isomerization is not a prerequisite for conformational changes to occur in the protein on the microsecond time scale.…”

“…In fact, in an early alternative hypothesis (11), the essential process was proposed to be dielectric relaxation of the protein as a response to sudden polarization upon retinal excitation (11)(12)(13). Recent molecular dynamics calculations (14) and experiments (15,16) support this view.…”

Spatio-temporal hierarchy in the dynamics of a minimalist protein model

“…Energy flow, 1, 2 such as anisotropic heat diffusion through protein residues, or vibrational energy transfer through the vibrational states of a protein, has long motivated experimental and computational studies toward its role in photosensing, [3][4][5][6] photosynthesis, 7 ligand-binding and dissociation, [8][9][10][11][12][13] and allostery. [14][15][16] For example, the existence of particular pathways of anisotropic heat diffusion was proposed to explain the allostery of the proteins belonging to the PDZ domain family.…”