2H NMR lineshapes of immobilized uniaxially oriented membrane proteins (original) (raw)
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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, 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
Biochemistry, 1998
The orientation of prosthetic groups in membrane proteins is of considerable importance in understanding their functional role in energy conversion, signal transduction, and ion transport. In this work, the orientation of the retinylidene chromophore of bacteriorhodopsin (bR) was investigated using 2 H NMR spectroscopy. Bacteriorhodopsin was regenerated with all-trans-retinal stereospecifically deuterated in one of the geminal methyl groups on C 1 of the cyclohexene ring. A highly oriented sample, which is needed to obtain individual bond orientations from 2 H NMR, was prepared by forming hydrated lamellar films of purple membranes on glass slides. A Monte Carlo method was developed to accurately simulate the 2 H NMR line shape due to the distribution of bond angles and the orientational disorder of the membranes. The number of free parameters in the line shape simulation was reduced by independent measurements of the intrinsic line width (1.6 kHz from T 2e experiments) and the effective quadrupolar coupling constant (38.8-39.8 kHz from analysis of the line shape of a powder-type sample). The angle between the C 1 -(1R)-1-CD 3 bond and the purple membrane normal was determined with high accuracy from the simultaneous analysis of a series of 2 H NMR spectra recorded at different inclinations of the uniaxially oriented sample in the magnetic field at 20 and -50°C. The value of 68.7 ( 2.0°in darkadapted bR was used, together with the previously determined angle of the C 5 -CD 3 bond, to calculate the possible orientations of the cyclohexene ring in the membrane. The solutions obtained from 2 H NMR were then combined with additional constraints from linear dichroism and electron cryomicroscopy to obtain the allowed orientations of retinal in the noncentrosymmetric membrane structure. The combined data indicate that the methyl groups on the polyene chain point toward the cytoplasmic side of the membrane and the N-H bond of the Schiff base to the extracellular side, i.e., toward the side of proton release in the pump pathway. † Work supported by grants from the National Institutes of Health (GM 53484 to M.P.H., EY 10622 and EY 12049 to M.F.B., and GM 36564 to K.N.) and a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to S.M.). a The dihedral angles φd correspond to the different ring pucker conformations. The following definitions apply. The polyene chain vector connects C5 with the Schiff base nitrogen. c is the angle between the C5-Me bond and the polyene chain vector. The chain tilt MN is the angle between the polyene chain vector and the membrane normal, where the ring plane roll RMN is a rotation around this vector. Angles in parentheses correspond to the C5-C15 vector as a reference direction.
Biochemistry, 2000
The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at g1.55 Å resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 Å. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with 15 N and metabolically incorporated retinal, 13 C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in darkadapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-13 C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 Å from [indole-15 N]Trp86, this distance is 3.9 Å in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution. † The work of D.O. was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 533).
Bacteriorhodopsin: the mechanism of 2D-array formation and the structure of retinal in the protein
Biophysical Chemistry, 1995
Bacteriorhodopsin, the light driven proton pump of the extreme halophilic bacterium H. salinarium, is an integral membrane protein (M(r) ca. 26000) which forms 2D arrays in the purple membrane of the bacterium. It is this feature which has permitted the use of electron diffraction methods to resolve the protein structure to some degree of atomic detail, although the prosthetic group has not been fully resolved. However, the features which induce the protein to form these arrays have not been previously clarified. We have now shown that the protein array formation is driven by specific interaction of the protein with the charged phospholipid, phosphatidyl glycerol phosphate (or the sulphate derivative), a major (ca. 60%) lipid of the bacterial host membrane. In addition, in an effort to provide further structural information about the chromophore, retinal, of this protein, the orientation of the individual methyl groups of retinal have been determined from solid state deuterium NMR studies of the deuterated chromophore when in the protein binding site. This approach to structural resolution of the prosthetic group is ab initio, agrees with other studies on the chromophore and resolves new features of the bound retinal to a high degree (+/- 2 degrees) of precision. Here, these two studies on this integral membrane protein will be reviewed.
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.).
Biophysical Chemistry, 1995
From our earlier extensive protein-lipid reconstitution studies, the conditions under which bacteriorhodopsin forms organised 2D arrays in large unilamellar vesicles have been established using freeze-fracture electron microscopy. In a background bilayer matrix of phosphatidylcholine (diC,,,o), the protein can form arrays only when the anionic purple membrane lipid, phosphatidylglycerol phosphate (or the sulphate derivative) is present. Here we have now extended this work to investigate the effect of bilayer thickness on array formation. Phosphatidylcholines with various chain lengths (diC,,:,, diCIko and diC,& and which form bilayers of well defined bilayer thickness, have been used as the matrix into which bacteriorhodopsin, together with minimal levels (c. 4-10 lipids per bacteriorhodopsin) of diphytanyl phosphatidylglycerol phosphate, has been reconstituted. Arrays are formed in all complexes and bilayer thickness appears only to alter the type of array formed, either as an orthogonal or as an hexagonal array.
Communications biology, 2023
The K intermediate of proton pumping bacteriorhodopsin is the first intermediate generated after isomerization of retinal to the 13-cis form. Although various structures have been reported for the K intermediate until now, these differ from each other, especially in terms of the conformation of the retinal chromophore and its interaction with surrounding residues. We report here an accurate X-ray crystallographic analysis of the K structure. The polyene chain of 13-cis retinal is observed to be S-shaped. The side chain of Lys216, which is covalently bound to retinal via the Schiff-base linkage, interacts with residues, Asp85 and Thr89. In addition, the Nζ-H of the protonated Schiff-base linkage interacts with a residue, Asp212 and a water molecule, W402. Based on quantum chemical calculations for this K structure, we examine the stabilizing factors of distorted conformation of retinal and propose a relaxation manner to the next L intermediate.
Transmembrane location of retinal in bacteriorhodopsin by neutron diffraction
Biochemistry, 1990
The transmembrane location of the chromophore of bacteriorhodopsin was obtained by neutron diffraction on oriented stacks of purple membranes. Two selectively deuterated retinals were synthesized and incorporated in bacteriorhodopsin by using the retinal-mutant JW5: retinal-d,, (D1 1) contained 11 deuterons in the cyclohexene ring, and retinal-d5 (D5) had 5 deuterons as close as possible to the Schiff base end of the chromophore. The membrane stacks had a lamellar spacing of 53.1 A at 86% relative humidity. Five orders were observed in the lamellar diffraction pattern of the D11, D5, and nondeuterated reference samples. The reflections were phased by D20-H20 exchange. The absolute values of the structure factors were nonlinear functions of the D 2 0 content, suggesting that the coherently scattering domains consisted of asymmetric membrane stacks. The centers of deuteration were determined from the observed intensity differences between labeled and unlabeled samples by using model calculations and Fourier difference methods. With the origin of the coordinate system defined midway between consecutive intermembrane water layers, the coordinates of the center of deuteration of the D11 and D5 label are 10.5 f 1.2 and 3.8 f 1.5 A, respectively. Alternatively, the label distance may be measured from the nearest membrane surface as defined by the maximum in the neutron scattering length density at the water/membrane interface. With respect to this point, the D11 and D5 labels are located at a depth of 9.9 f 1.2 and 16.6 f 1.5 A, respectively.
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.