Chromophore Orientation in Bacteriorhodopsin Determined from the Angular Dependence of Deuterium Nuclear Magnetic Resonance Spectra of Oriented Purple Membranes † (original) (raw)
Related papers
Biochemistry, 1992
The orientation and conformation of retinal within bacteriorhodopsin of the purple membrane of Halobacterium halobium was established by solid-state deuterium N M R spectroscopy, through the determination of individual chemical bond vectors. The chromophore ([2,4,4,16,16,16,17,17,17,18,18-2H1 I]retinal) was specifically deuterium-labeled on the cyclohexene ring and incorporated into the protein. Auniaxially oriented sample of purple membrane patches was prepared and measured at a series of inclinations relative to the spectrometer field. 31P N M R was used to characterize the mosaic spread of the oriented sample, and computer simulations were applied in the analysis of the *H N M R and 31P N M R spectral line shapes. From the deuterium quadrupole splittings, the specific orientations of the three labeled methyl groups on the cyclohexene ring could be calculated. The two adjacent methyl groups (on CI) of the retinal were found to lie approximately horizontal in the membrane and make respective angles of 94" f 2" and 7 5 O f 2 O with the membrane normal. The third group (on C,) points toward the cytoplasmic side with
2H NMR lineshapes of immobilized uniaxially oriented membrane proteins
Solid State Nuclear Magnetic Resonance, 1993
As a method for the structure determination of integral membrane proteins or other large macromolecular complexes, a solid state 'H NMR approach is presented, capable of measuring the orientations of individual chemical bond vectors. In an immobilized uniaxially oriented sample, the bond angle of a deuterium-labelled methyl group relative to the axis of ordering can be calculated from the quadrupole splitting in the "zero-tilt" spectrum where the sample normal is aligned parallel to the spectrometer field direction. However, since positive and negative values of this splitting cannot be distinguished, there may appear to be two solutions, of which only one describes the correct molecular geometry. We show that it is possible to determine the bond angle uniquely between 0" and 90", by analysing the lineshapes of a tilt series of spectra acquired over different sample inclinations. The lineshape equation describing such oriented 'H NMR spectra will be derived (for asymmetry parameter TJ = 0) and discussed, with an illustration of the various linebroadening effects from which the orientational distribution function in the macroscopically ordered system can be determined. This strategy is then applied to specifically deuterium-labelled retinal in dark-adapted bacteriorhodopsin, prepared in a uniaxially oriented sample from purple membrane fragments. From the quadrupole splitting in the zero-tilt spectrum and by lineshape simulations, the deuteromethyl group at C,, on retinal is found to make an angle of 32"k 1" with the membrane normal, and the sample mosaic spread to be around f8". The resulting orientation of retinal is in excellent agreement with its known structure in bacteriorhodopsin, and together with the results on other methyl groups it will be possible to construct a detailed picture of the chromophore in the protein binding pocket.
Distorted Structure of the Retinal Chromophore in Bacteriorhodopsin Resolved by 2H-NMR
Biochemistry, 1994
Structural details about the geometry of the retinal chromophore in the binding pocket of bacteriorhodopsin are revealed by measuring the orientations of its individual methyl groups. Solid-state 2H-NMR measurements were performed on macroscopically oriented samples of purple membrane patches, containing retinal specifically deuterium-labeled at one of the three methyl groups along the polyene chain (Clg, C19, C20). The deuterium quadrupole splitting of each "zero-tilt" spectrum is used to calculate the orientation of the corresponding C-CD3 bond vector with respect to the membrane normal; however, two possible solutions may arise. These ambiguities in angle could be resolved by recording a tilt series of spectra at different sample inclinations to the magnetic field and analyzing the resulting complex line shapes with the aid of computer simulations. The angles for the C18, C19, and C20 group are found to be 37 f l o , 40 f 1 O , and 32 i 1 O , respectively. These highly accurate values imply that the polyene chain of the retinal chromophore is not straight but rather has an in-plane curvature and possibly an out-of-plane twist. Together with the angles of the remaining methyl groups on the cyclohexene ring that have been measured previously, an overall picture has thus emerged of the intramolecular conformation and the three-dimensional orientation of retinal within bacteriorhodopsin. The deduced geometry confirms and refines the known structural information on the chromophore, suggesting that this 2H-NMR strategy may serve as a valuable tool for other membrane proteins. Abstract published in Advance ACS Abstracts, April 15, 1994. Abbreviations: 2H-NMR, deuterium nuclear magnetic resonance; BR, bacteriorhodopsin; PM, purple membrane; AUQ, quadrupole splitting.
Biochemistry, 1999
The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR ( 2 H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C 1 or C 5 of the -ionone ring or carbon C 9 of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH. 2 H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-syn and all-trans, 15-anti chromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at -50°C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5°C and then rapidly cooled to -50°C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the 2 H NMR line shapes, the angles between the individual C-CD 3 bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark-and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C 5 -C 13 part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C 9 -CD 3 bond shows the largest orientational change of 7°in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C 9 -methyl group and Trp 182 in helix F. † Work supported by grants from the National Institutes of Health (GM 53484 to M.P.H., EY 12049 to M.F.B., and GM 36564 to K.N.) and a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to S.M.).
Solid-State 2H NMR spectroscopy of retinal proteins in aligned membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007
Solid-state 2 H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2 H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C 2 H 3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2 H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark-and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2 H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2 H NMR is thus illuminating in terms of their different biological functions.
Macroscopic Orientation of Natural and Model Membranes for Structural Studies
Analytical Biochemistry, 1997
recent years solid-state NMR spectroscopy (3, 4) on One approach for obtaining high-resolution strucmacroscopically oriented lipid bilayers has rapidly tural and functional information for biomembranes emerged as an alternative approach to elucidate strucand their proteins is by static solid-state NMR of oritural and functional features of membrane-bound pepented systems. Here, a general procedure to align fully tides and proteins. In such aligned systems, lipids and functional biological membranes containing large proteins are arranged uniaxially around the memmembrane proteins (M r ú30,000) is described. The brane, allowing normal orientation of the molecule method, based on the isopotential spin-dry ultracenbackbone relative to the substratum. In combination trifugation technique, relies on the centrifugation of with isotopic labeling (e.g., 2 H, 13 C, 15 N) this NMR apmembrane fragments onto a support with simultaneproach has successfully been used to determine the ous, or subsequent, partial evaporation of the solvent complete secondary structure of the M2 channel pepwhich aids alignment. The quality of orientation, as tide and fd coat protein (3), while the orientation of shown by the mosaic spread of the samples, was monithe antibiotic peptide magainin has been resolved in tored by static solid-state 31 P NMR for the phospholipbilayers (5). The complete secondary structure of gramids and by 2 H NMR for a deuterated retinal in bovine icidin and its dynamic properties in membranes have rhodopsin. The generality of this method is demonalso been obtained using 2 H and 15 N NMR (6-9). In strated with three different membranes containing boaddition, the complete structure and orientation of deuvine rhodopsin in reconstituted bilayers, natural terated retinal in bacteriorhodopsin at different states membranes with the red cell anion exchange transport protein in erythrocytes, band 3, and the nicotinic ace-of its photocycle has been resolved (10). The average tylcholine receptor.
FEBS Letters, 1998
Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2 H NMR approach was used. Bovine rhodopsin containing 11-cis retinal, specifically deuterated at its methyl groups at the C 19 or C 20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31 P NMR. Analysis of the obtained 2 H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.
Photochemistry and Photobiology, 1992
We have determined the transition dipole moment orientation of the chromophore during the photocycle of bacteriorhodopsin by photoselection and time-resolved linear dichroism experiments with samples of oriented immobilized purple membranes. This technique offers two important advantages over experiments with isotropic aqueous suspensions: (1) the depolarization due to the rotational diffusion of the membranes is eliminated, (2) the sensitivity for detecting the orientation of the transition dipole moment of intermediates is greatly increased. The appropriate equations for the analysis of time-resolved linear dichroism experiments with samples of oriented immobilized membranes will be presented. In the transition from the ground state of bacteriorhodopsin to the Mintermediate, the transition dipole moment tilts out of the plane of the membrane by about 3". On the basis of current structural information on the plane of the chromophore and the orientation of its C(19) and C(20) methyl groups, a tilt of the transition dipole moment into the plane of the membrane would have been expected if it is assumed that the orientation of the conjugated polyene chain from C(5) to C(13) is the same in both states. The experimental result may be explained by an 11" tilt of the C(5) to C(13) part of the chain out of the plane of the membrane with the C(20) methyl group moving towards the cytoplasmic side of the membrane by about 1.7 d; and the cyclohexene ring staying fixed. This interpretation is supported by recent neutron diffraction experiments on the chromophore position in the M-intermediate.
Journal of the American Chemical Society, 1999
We present the first application of MAOSS (magic angle oriented sample spinning) NMR spectroscopy to a large membrane protein. This new solid-state NMR approach is used to study the orientation of the deuterated methyl group in [18-CD 3 ]-retinal in oriented bacteriorhodopsin in both the photocycle ground state (bR 568 ) and in the photo intermediate state M 412 . Deuterium MAS spectra consist of a set of narrow spinning sidebands if the sample spinning rate does not exceed the anisotropy of the quadrupole interaction. In ordered systems, such as proteins in oriented membranes, each sideband intensity is orientationally dependent. The observed MAS sideband pattern is modulated in a highly sensitive way by changes in the molecular orientation of the CD 3 group during the transition from all-trans-to 13-cis-retinal upon photoactivation. The significant improvement in spectral sensitivity and resolution, compared to static NMR on oriented samples, allows a reliable and precise data analysis even from lower spin concentrations and has more general consequences for studying oriented membrane proteins by NMR. MAOSS NMR is shown to be a feasible method for the accurate determination of local molecular orientations in large molecular systems which are currently a challenge for crystallography.