Refinement of the Geometry of the Retinal Binding Pocket in Dark-Adapted Bacteriorhodopsin by Heteronuclear Solid-State NMR Distance Measurements † (original) (raw)
Related papers
Biophysical Journal, 1997
A 3D model of the transmembrane 7-a-bundle of rhodopsin-like G-protein-coupled receptors (GPCRs) was calculated using an iterative distance geometry refinement with an evolving system of hydrogen bonds, formed by intramembrane polar side chains in various proteins of the family and collectively applied as distance constraints. The a-bundle structure thus obtained provides H bonding of nearly all buried polar side chains simultaneously in the 410 GPCRs considered. Forty evolutionarily conserved GPCR residues form a single continuous domain, with an aliphatic "core" surrounded by six clusters of polar and aromatic side chains. The 7-a-bundle of a specific GPCR can be calculated using its own set of H bonds as distance constraints and the common "average" model to restrain positions of the helices. The bovine rhodopsin model thus determined is closely packed, but has a few small polar cavities, presumably filled by water, and has a binding pocket that is complementary to 1 1-cis (6-s-cis, 1 2-s-trans, C=N ant/)-retinal or to all-trans-retinal, depending on conformations of the Lys296 and Trp265 side chains. A suggested mechanism of rhodopsin photoactivation, triggered by the cis-trans isomerization of retinal, involves rotations of Glu134, Tyr223, Trp265, Lys296, and Tyr306 side chains and rearrangement of their H bonds. The model is in agreement with published electron cryomicroscopy, mutagenesis, chemical modification, cross-linking, Fourier transform infrared spectroscopy, Raman spectroscopy, electron paramagnetic resonance spectroscopy, NMR, and optical spectroscopy data. The rhodopsin model and the published structure of bacteriorhodopsin have very similar retinal-binding pockets.
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, 2004
Proton pumps utilize a chemical or photochemical reaction to create pH and electrical gradients between the interior and the exterior of cells and organelles that energize ATP synthesis and the accumulation and extrusion of solutes and ions. G-protein coupled receptors bind agonists and assume signaling states that communicate with the coupled transducers. How these two kinds of proteins convert chemical potential to a proton transmembrane electrochemical potential or a signal are the great questions in structural membrane biology, and they may have a common answer. Bacteriorhodopsin, a particularly simple integral membrane protein, functions as a proton pump but has a heptahelical structure like membrane receptors. Crystallographic structures are now available for all of the intermediates of the bacteriorhodopsin transport cycle, and they describe the proton translocation mechanism, step by step and in atomic detail. The results show how local conformational changes propagate upon the gradual relaxation of the initially twisted photoisomerized retinal toward the two membrane surfaces. Such local-global conformational coupling between the ligand-binding site and the distant regions of the protein may be the shared mechanism of ion pumps and G-protein related receptors.
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.
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.
Biochemistry, 2001
In the absence of a high-resolution diffraction structure, the orientation and conformation of the protonated Schiffs base retinylidinium chromophore of rhodopsin within the opsin matrix has been the subject of much speculation. There have been two recent reliable and precise NMR results that bear on this issue. One involves a determination of the C20-C10 and C20-C11 distances by Verdegem et al. [Biochemistry 38, 11316-11324 (1999)]. The other is the determination of the orientation of the methine C to methyl group vectors C5-C18, C9-C19, and C13-C20 relative to the membrane normal by Gröbner et al. [Nature 405 (6788), 810-813 ]. Using molecular orbital methods that include extensive configuration interaction, we have determined what we propose to be the minimum energy conformation of this chromophore. The above NMR results permit us to check this structure in the C10-C11dC12-C13 region and then to check the global structure via the relative orientation of the three C18, C19, and C20 methyl groups. This method provides a detailed structure and also the orientation for the retinyl chromophore relative to the membrane normal and argues strongly that the protein does not appreciably alter the chromophore geometry from its minimum energy configuration that is nearly planar s-trans at the 6-7 bond. Finally, the chromophore structure and orientation presented in the recently published X-ray diffraction structure is compared with our proposed structure and with the deuterium NMR results. .
Investigation of the Binding Geometry of a Peripheral Membrane Protein †
Biochemistry, 2005
A growing number of modules including FYVE domains target key signaling proteins to membranes through specific recognition of lipid headgroups and hydrophobic insertion into bilayers. Despite the critical role of membrane insertion in the function of these modules, the structural mechanism of membrane docking and penetration remains unclear. In particular, the three dimensional orientation of the inserted proteins with respect to the membrane surface is difficult to define quantitatively. Here, we determined the geometry of the micelle penetration of early endosome antigen 1 (EEA1) FYVE domain by obtaining NMR-derived restraints that correlate with the distances between protein backbone amides and spin labeled probes. The 5-and 14-doxyl-phosphatidylcholine spin labels were incorporated into dodecylphosphocholine (DPC) micelles, and the reduction of amide signal intensities of the FYVE domain due to paramagnetic relaxation enhancement was measured. The vector of the FYVE domain insertion was estimated relative to the molecular axis by minimizing the paramagnetic restraints obtained in phosphatidylinositol 3-phosphate (PI3P)-enriched micelles containing only DPC or mixed with phosphatidylserine (PS). Additional distance restraints were obtained using a novel spin label mimetic of PI(3)P that contains a nitroxyl radical near the threitol group of the lipid. Conformational changes indicative of elongation of the membrane insertion loop (MIL) were detected upon micelle interaction, in which the hydrophobic residues of the loop tend to move deeper into the non-polar core of micelles. The micelle insertion mechanism of the FYVE domain defined in this study is consistent with mutagenesis data and chemical shift perturbations and demonstrates the advantage of using the spin label NMR approach for investigating the binding geometry by peripheral membrane proteins.
Biochemistry, 2001
C 10 ]Retinal prepared by total synthesis is reconstituted with opsin to form rhodopsin in the natural lipid membrane environment. The 13 C shifts are assigned with magic angle spinning NMR dipolar correlation spectroscopy in a single experiment and compared with data of singly labeled retinylidene ligands in detergent-solubilized rhodopsin. The use of multispin labeling in combination with 2-D correlation spectroscopy improves the relative accuracy of the shift measurements. We have used the chemical shift data to analyze the electronic structure of the retinylidene ligand at three levels of understanding: (i) by specifying interactions between the 13 C-labeled ligand and the G-protein-coupled receptor target, (ii) by making a charge assessment of the protonation of the Schiff base in rhodopsin, and (iii) by evaluating the total charge on the carbons of the retinylidene chromophore. In this way it is shown that a conjugation defect is the predominant ground-state property governing the molecular electronics of the retinylidene chromophore in rhodopsin. The cumulative chemical shifts at the odd-numbered carbons (∆σ odd) of 11-Z-protonated Schiff base models relative to the unprotonated Schiff base can be used to measure the extent of delocalization of positive charge into the polyene. For a series of 11-Z-protonated Schiff base models and rhodopsin, ∆σ odd appears to correlate linearly with the frequency of maximum visible absorption. Since rhodopsin has the largest value of ∆σ odd , the data contribute to existing and converging spectroscopic evidence for a complex counterion stabilizing the protonated Schiff base in the binding pocket.
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.