Functional electric field changes in photoactivated proteins revealed by ultrafast Stark spectroscopy of the Trp residues

Abstract

retinal proteins ͉ dipolar interactions ͉ tryptophan ͉ ultraviolet probe T he membrane protein bacteriorhodopsin (bR) is a proton pump, the biological activity of which is triggered by the absorption of light by the protonated retinal cofactor (1, 2). On light excitation, the retinal moiety undergoes an isomerization from 13-cis to all-trans that proceeds with very high speed (Ϸ0.5 ps) and large quantum efficiency (Ϸ0.64). In the excited Franck-Condon state, before isomerization, a large photoinduced charge translocation along retinal increases its dipole moment by as much as 10-30 D (3, 4). Remarkably, the photoisomerization of the same chromophore in any environment other than that of the natural protein scaffold is no longer specific and it is less efficient and slower (5), indicating the enzymatic role of the protein environment that enhances the speed and selectivity of the retinal isomerization. Site-directed mutagenesis has shown that charged amino acid residues closely interacting with the charged retinal moiety (Arg 82 , Asp 85 , Asp 212 ) largely influence the isomerization speed (6, 7). Theoretical investigations of a retinal-like model compound (8) rationalize these findings in modeling how a chloride counterion affects the energetics and photoreactivity. In addition, within the protein, the large photoinduced charge translocation along retinal is expected to induce a strong, ultrafast dielectric response of the full protein environment (9, 10), which could also participate in the enzymatic function by driving specifically the isomerization path to all-trans retinal. Besides, the exact nature of the driving force for the biological activity of the protein has long been (11) and still is a subject of intensive investigations. Hence, protein conformational changes similar to those observed in functional wild-type (WT) bR have been observed in artificial proteins with nonisomerizing retinal chromophores (12), suggesting that the initial charge translocation alone would activate the modified proteins. In the initial steps of the photocycle, the protein has to convert and store light energy, so as to drive the primary proton transfer from the protonated retinal to the Asp 85 counter ion, and successive proton transfer reactions via small conformational rearrangements of the protein. Several origins have been proposed and debated for the dominating driving force leading to the primary and subsequent proton transfers, among which are (i) energy storage in the strained geometry of the isomerized chromophore (13), and (ii) electrostatic energy storage (14), either mainly due to the modification by the isomerization of the electrostatic interactions between the protein and the donor/ acceptor pair (15), or to a light-induced charge separation between primary proton donor and acceptor (16).To investigate the light-induced electrostatic and/or dielectric modifications along or in the vicinity of retinal, we performed ultrafast absorption spectroscopy on the nearby tryptophan residues. Wild-type bR contains 8 try...

Aborted double bicycle-pedal isomerization with hydrogen bond breaking is the primary event of bacteriorhodopsin proton pumping

“…The structural evolution described above seems to be consistent with the photoinduced changes in permanent dipole moment of the chromophore that have been experimentally monitored by Léonard et al (19). In particular, the recorded transient response (i.e., modifications in UV-absorption spectrum) of the nearby Trp86 residue has been used as a probe of the ultrafast changes in the local PSBT charge distribution (i.e., the dipole moment variation-Δμ-with respect to the one recorded in ground state bR).…”

“…Structural parameters optimized for the mobile W402 setup are reported in italics when differing from those pertaining to the fixed W402 setup (see Computational Methods and SI Text). stants that match S 0 recovery and photoproduct K formation, although the initial bR value is not fully recovered (19), calling for a change in the local electrostatic environment that remains long after isomerization. This trend is consistent with the small value of Δμ calculated at K consistently with a charge distribution similar to that found in bR.…”

“…Such a barrier would explain the 450-fs short-lived quasistationary state observed by Ruhman et al (5) that is assigned to the fluorescent state I (16)(17)(18) and precedes decay to the ground state (S 0 ). Recent experiments by Léonard et al (19) have also revealed that the photoinduced changes in the permanent dipole moment of PSBT are mirrored by the absorption of Trp86 up to the first stable (i.e., cryogenically trapped) photoproduct K (7). Relative to bR, K bears 11.7-15.9 kcal mol −1 stored photon energy (20).…”

Terahertz Absorption Spectroscopy of a Liquid Using a Polarity Probe: A Case Study of Trehalose/Water Mixtures

“…[5,19,20] In the case of MQ, S 0 !S 1 excitation at 400 nm reduces the dipole moment m from 10.8 to 5.8 D, [21] and the local electric field is switched down instantaneously. As the new field acts on nearby groups with partial charges, these reorient and collectively create the reaction field R(t) (Figure 2 b).…”

The feasibility of coherent energy transfer in microtubules

“…Exciton stick spectra for absorption, CD and linear dichroism (LD) were calculated from the orientations of the Trp molecules in the dominant confirmation and the diagonalized Hamiltonian matrix using the formula described by Pearlstein [73]. Experimental spectra were calculated by applying a Gaussian function to each exciton transition using an assumed full-width half maximum value of 3500 cm 21 as commonly observed for Trp [74,75]. These were then compared with experimental absorption, CD and LD spectra adapted from Mozo-Villarias et al [76], Clark et al [77] and Marrington et al [78], respectively.…”