Structure determination of the cyclohexene ring of retinal in bacteriorhodopsin by solid-state deuterium NMR (original) (raw)
Related papers
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.
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.
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.
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.
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.
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.
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.).
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.
Biophysical Journal, 1989
The absolute direction of nique. Compared with the known points away from the cytoplasmic surthe retinal chromophore of bacterio-adsorbed geometry of free retinylidene face of the purple membrane. The rhodopsin relative to the membrane Schiff base on a glass substrate, our implication of this finding is discussed in plane is investigated by using an optical data indicate the 3-ionone ring of the light of other chemical and structural second-harmonic interference tech-chromophore of bacteriorhodopsin results on bacteriorhodopsin.