Karim Fahmy

Photoisomerization in bacteriorhodopsin studied by FTIR, linear dichroism and photoselection experiments combined with quantum chemical theoretical analysis

Abstract Orientations of IR transition moments of the retinal chromophore of bacteriorhodopsin (BR) and of the apo-protein are investigated by FTIR linear dichroism and photoselection measurements. Low temperature difference spectra for the photoinduced transitions of BR to its photocycle intermediates K and L are evaluated using improved methods. Quantum chemical calculations of directions of IR and electronic transition moments of model chromophores are employed to analyze corresponding observations. The chromophore of light-adapted BR 568 is shown to exhibit small (15–30°) twists around the Cue5f8C single bonds of retinals polyene chain but no large overall helicity (⩽15°). The average retinal plane is demonstrated to form an angle of 90±20° with the plane of the purple membrane. The C 9 ue5fbC 10 double bond of retinal is found approximately parallel to the plane of the membrane. Upon photoisomerization the orientation of the chromophore moiety from C 1 to C 13 is estimated to be largely conserved. The single bond twists of the chromophore in L are shown to be larger than those in BR 568 . This result is in agreement with the previous prediction of increased single bond twists in L, which can cause a p K decrease of the chromophore and, thereby, enforce its deprotonation in the L→M transition [Schulten and Tavan, Nature, 272 (1978) 85].

Photoactivated state of rhodopsin and how it can form

A variety of spectroscopic and biochemical studies of the photoreceptor rhodopsin have revealed conformation changes which occur upon its photoactivation. Assignment of these molecular alterations to specific regions in the receptor has been attempted by studying native opsin regenerated with synthetic retinal analogs or recombinant opsins regenerated with 11-cis retinal. We propose a model for the photoactivation mechanism which defines off and on states for individual molecular groups. These groups have been identified to undergo structural alterations during photoactivation. Analysis of mutant pigments in which specific groups are locked into their respective on or off states provides a framework to identify determinants of the active conformation as well as the minimal number of intramolecular transitions to switch to this conformation. The simple model proposed for the active-state of rhodopsin can be compared to structural models of its ground-state to localize chromophore-protein interactions that may be important in the photoactivation mechanism.

Binding of Transducin and Transducin-Derived Peptides to Rhodopsin Studied by Attenuated Total Reflection–Fourier Transform Infrared Difference Spectroscopy

Fourier transform infrared difference spectroscopy combined with the attenuated total reflection technique allows the monitoring of the association of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation of the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominent absorption increases occur at 1735 cm-1, 1640 cm-1, 1550 cm-1, and 1517 cm-1. The D2O sensitivity of the broad carbonyl stretching band around 1735 cm-1 indicates that a carboxylic acid group becomes protonated upon formation of the activated complex. Reconstitution of rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas pH affects the intensity of the carbonyl stretching band. AC-terminal peptide comprising amino acids 340-350 of the transducin alpha-subunit reproduces the frequencies and isotope sensitivities of several of the transducin-induced bands between 1500 and 1800 cm-1, whereas an N-terminal peptide (aa 8-23) does not. Therefore, the transducin-induced absorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm-1 vibration is also seen in the complex with C-terminal peptides devoid of free carboxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin.

IDENTIFICATION OF THE PROTON ACCEPTOR OF SCHIFF BASE DEPROTONATION IN BACTERIORHODOPSIN: A FOURIER-TRANSFORM-INFRARED STUDY OF THE MUTANT ASP85 GLU IN ITS NATURAL LIPID ENVIRONMENT

Abstract— In order to assign the proton acceptor for Schiff base deprotonation in bacteriorhodopsin to a specific Asp residue, the photoreaction of the Asp85 → Glu mutant, as expressed in Halobacterium sp. GRB, was investigated by static low‐temperature and time‐resolved infrared difference spec‐troscopy. Measurements were also performed on the mutant protein labeled with [4‐13C]Asp which allowed discrimination between Asp and Glu residues. 14,15‐di13C‐retinal was incorporated to distinguish amide‐II absorbance changes from changes of the ethylenic mode of the chromophore. In agreement with earlier UV‐VIS measurements, our data show that from both the 540 and 610 nm species present in a pH‐dependent equilibrium, intermediates similar to K and L can be formed. The 14 ms time‐resolved spectrum of the 540 nm species shows that a glutamic acid becomes protonated in the M‐like intermediate, whereas the comparable difference spectrum of the 610 nm species demonstrates that in the initial state a glutamic acid is already protonated. In conjunction with earlier observations of protonation of an Asp residue in wild‐type M, the data provide direct evidence that the proton acceptor in the deprotonation reaction of the Schiff base is Asp85.

Structural investigation of bacteriorhodopsin and some of its photoproducts by polarized Fourier transform infrared spectroscopic methods-difference spectroscopy and photoselection.

The direction of selected IR-transition moments of the retinal chromophore of bacteriorhodopsin (BR) and functional active amino acid residues are determined for light- and dark-adapted BR and for the intermediates K and L of the photocycle. Torsions around single bonds of the chromophore are found to be present in all the investigated BR states. The number of twisted single bonds and the magnitude of these torsions decreases in the order K, L, light-adapted BR, dark-adapted BR. In the last, only the C(14)-C(15) single bond is twisted. The orientation of molecular planes and chemical bonds of such protein side chains, which are perturbed during the transition of light-adapted BR to the respective intermediates, are deduced and the results compared with the current three dimensional model of BR. Trp 86 and Trp 185 are found to form a rigid part of the protein, whereas Asp 96 and Asp 115 perform molecular rearrangements upon formation of the L-intermediate.

THE PHOTOREACTION OF THE DEIONIZED FORM OF THE PURPLE MEMBRANE INVESTIGATED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— Deionization of the purple membrane of Halobacterium halobium shifts the visible absorption maximum from 570 to 605 nm and inhibits proton transport. FTIR‐difference‐spectra of this blue membrane at 280 K reveal that the retinal chromophore adopts a 13‐cis and all‐trans geometry in a light dependent ratio. In contrast to purple membrane the 13‐cu isomer forms much faster in the dark. The all‐trans component produces an L‐intermediate which can be stabilized at 170 K. Spectral characteristics are similar to normal L. including comparable changes of internal aspartic acids of the opsin. However, stronger changes in the amide‐I absorption are observed. IR bands of the chromo‐protein states are assigned to retinal normal modes by the use of bacteriorhodopsin regenerated withC‐labeled retinals.

Biomolecular interactions studied by FT-IR-ATR spectroscopy

FTIR-spectroscopy in combination with the attenuated total reflection (ATR)-technique allows the monitoring of binding reactions between molecules over a large range of molecular masses [1, 2]. In this study an extension to this method is described in which the ligand can be added to immobilized molecules by dialysis; thereby, preventing mechanical perturbations at the ATR-crystal surface often encountered with flow injection methods. To demonstrate the broad field of applications the binding of a heterotrimeric G-protein transducin with its membrane bound receptor rhodopsin was investigated as well as the pH-induced conformational change of the photoactivated receptor.

Binding of Transducin and Transducin-Derived Peptides to Rhodopsin Studied by ATR-FTIR Spectroscopy

Fourier-transform-infrared (FTIR) difference spectroscopy combined with site-directed mutagenesis has identified vibrational changes of membrane-embedded amino acid side chains during formation of the metarhodopsin II (MII) photoproduct of the photoreceptor rhodopsin (rho) [1–2]. In contrast, little is known about vibrational difference bands that can be attributed to changes on the cytoplasmic surface of rho interacting with its cognate G protein transducin (td). In the present study, attenuated total reflection (ATR) FTIR difference spectroscopy has been employed to monitor rho td interactions in the presence of bulk water as compared to transmissive spectroscopy [3]. Dark binding of td to rho [4] transfers protein from the aqueous phase into the evanescent field of an internal reflection element to which rho has been adsorbed. Subsequent illumination causes absorption changes representative for the activation of the preformed rhodopsin td complex. The absorption changes evoked by the presence of td are generated by subtracting a MII difference spectrum obtained in the absence of td from that in the presence of td under otherwise identical conditions resulting in the spectrum shown in Fig. 1.. Difference bands at 1693, 1662 (negative) and 1640 cm−1 (positive) are already present in MII difference spectra but are enhanced by the presence of td., therefore, they cause peaks even after spectral subtraction. This may indicate that these bands are caused by specific surface conformations of rhodopsin which become stabilized by td. Stabilization of a MIIb conformation as compared to a MIIa conformation in the absence of td is actually expected [5]. Absorption increases at frequencies different from those of rho and MIIa are induced by td at 1517, 1460, 1308, and at 1735 cm−1. The sensitivity of the latter band to H/D exchange (not shown) suggests that part of this broad feature is due to the C=O stretching mode of a carboxylic acid that becomes protonated in the rho td complex. Glu134 of rho is a possible candidate for this carboxylic acid [6–8]. A peptide comprising the 11 C-terminal amino acids of the td α subunit also induces the 1640 cm−1 absorption increase as well as the D2O-sensitive band at 1735 cm−1. Therefore, these absorption changes seem to be specific for the interaction of rho with the C terminus of td α-subunit previously reported to be implicated in rho binding [91.

Molecular Determinants of the Active Conformation of Rhodopsin Studied by Attenuated Total Reflectance FTIR Difference Spectroscopy

The vertebrate photoreceptor rhodopsin is a member of the super family of G protein-coupled receptors (GPCR). The ease of light-dependent, as compared to ligand-dependent activation and the abundance by which rhodopsin can be prepared from bovine retinae have rendered this system an intensely studied model for GPCRs in general. We have used the attenuated total reflectance method to obtain FTIR difference spectra of the light-induced formation of the metarhodopsin I/metarhodopsin II photoproduct equilibrium at well defined pH and ionic strengths in aqueous solution. In contrast to previous measurements on hydrated films of rhodopsins in which MI and Mil had to be stabilized at -20°C and 5°C, respectively, the ATR technique allows to shift the MI/MII equilibrium at a fixed temperature by varying the pH while interferograms are being recorded. Therefore, it is possible to study conformational changes occuring with the MI to Mil transition, i.e., pH-induced difference spectra of the MI to MII transition can be obtained directly from a once illuminated sample. The advantage of this approach versus temperature stabilization is twofold: from previous experiments the MI/MII difference can only be estimated by subtraction of seperate light-induced rho to MI and rho to MII difference spectra obtained from independent samples. Therefore, absorption bands of dark rhodopsin are still present.