GPCRs: an update on structural approaches to drug discovery

G-protein-coupled receptors (GPCRs) are a major opportunity for drug discovery in the post-genomic era. There are thought to be more than 500 therapeutically relevant GPCRs out of a total of over 700 identified to date, although only one, rhodopsin, has been the subject of a full 3D X-ray crystallography study. Two structurally related proteins, bacteriorhodopsin and sensory rhodopsin, which are not GPCRs but are part of the seven-helix membrane receptor family, have also been the subject of X-ray crystallographic studies and have been used in GPCR modeling studies. The significant differences between these rhodopsin structures, the relatively low sequence homology between individual GPCRs, and some difficulties in rationalizing point-mutation data suggests that homology-based molecular modeling alone will not provide the accurate structural information on individual receptors required for ligand design and in silico screening. In the absence of such structural information, several approaches can be used to assist in the discovery of ligands.     

The similarity of the primary structure and homology of rhodopsin, beta-adrenoreceptor and muscarinic cholinoceptor

Computer analysis has been made of the primary structure of 6 different types of receptor proteins: rhodopsin, adrenoreceptor, muscarinic acetylcholine receptor, insulin receptor, nicotinic cholinoreceptor, and bacteriorhodopsin. The aim of the present investigation was to elucidate, at least partially, to what extent insignificant similarity in the primary structure of rhodopsin, muscarinic cholinoreceptor and adrenoreceptor is due to divergent, but not convergent, evolution. Nicotinic cholinoreceptor, bacteriorhodopsin and insulin receptor were chosen for comparison with rhodopsin, adrenoreceptor and muscarinic cholinoreceptor since each of these proteins exhibits this or that structural or functional property which is common for rhodopsin, adrenoreceptor or muscarinic cholinoreceptor; on the other hand, nicotinic cholinoreceptor, bacteriorhodopsin and insulin receptor differ from other receptor proteins by their molecular mechanisms. Comparison of the primary structure of rhodopsin, adrenoreceptor and muscarinic cholinoreceptor on the one hand, and insulin receptor, nicotinic cholinoreceptor and bacteriorhodopsin on the other indicates that only the former exhibit similar primary structure, whereas insulin receptor, nicotinic cholinoreceptor and bacteriorhodopsin show no similarity neither in their primary structure, nor in the primary structure of rhodopsin and other receptor proteins which are similar to the latter with respect to their mode of action. The data obtained indicate that similarity in the primary structure between rhodopsin, muscarinic cholinoreceptor and adrenoreceptor is a consequence of divergent, not convergent, evolution; in other words, these receptor proteins are homologous.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

Mass spectrometric analysis of cyanogen bromide fragments of integral membrane proteins at the picomole level: application to rhodopsin

Advances in time-of-flight mass spectrometry allow unit mass resolution of proteins and peptides up to about 6000 Da molecular weight. Identification of larger proteins and study of their posttranslational or experimental modifications by mass analysis is greatly enhanced by cleavage into smaller fragments. Most membrane proteins are difficult to mass analyze because of their high hydrophobicity, typical expression in low quantities, and because the detergents commonly used for solubilization may be deleterious to mass analysis. Cleavage with cyanogen bromide is beneficial for analysis of membrane proteins since the methionine cleavage sites are typically located in hydrophobic domains and cleavage at these points reduces the size of the hydrophobic fragments. Cyanogen bromide also gives high cleavage yields and introduces only volatile contaminants. Even after cleavage membrane proteins often contain fragments that are difficult to chromatograph. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is capable of analyzing complex mixtures without chromatography. We present a MALDI MS method that quickly and reliably identifies the cyanogen bromide fragments and posttranslational modifications of reduced and alkylated bovine rhodopsin from as little as 30 pmol of rhodopsin in detergent-solubilized retinal rod disk membranes, using 1-5 pmol of digest per sample. The amino acid sequences of some of the peptides in the digest were confirmed by post source decomposition MS analysis of the same samples. The method appears to be general and applicable to the analysis of membrane proteins and the protein composition of membrane preparations.     

Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity-purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse-phase, high-performance liquid chromatography at 55 degrees C with the detergent octyl-beta-glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N-terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.     

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or nycodenz-adjusted density of the hydrated detergent micelle

The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach.     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.     

Sensory rhodopsin II and bacteriorhodopsin: light activated helix F movement.

EPR spectroscopy in combination with site directed spin labeling (SDSL) has become a valuable tool for structural investigations as well as for kinetic studies on proteins. This method has been especially useful for membrane proteins in yielding structural and functional data. This information is not easily available from other techniques, like, e.g., X-ray crystallography or electron microscopy. In the first part of this two part review, the topology of the sensory rhodopsin II/transducer complex (NpSRII/NpHtrII) derived from EPR constraints is compared to that obtained from X-ray crystallography. In the second part, the helix F movement observed for both sensory rhodopsin and bacteriorhodopsin is evaluated and discussed in order to establish a common mechanism after photoreceptor activation.     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.     

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.     

用神经网络法预测膜蛋白RH和BR的二级结构

膜蛋白的结构预测目前还十分困难.本文以两个典型的膜蛋白为选材,用神经网络方法对其二级结构做了预测.预测结果与实验资料的符合率与该方法用于球蛋白时的结果一致,因此是较好的.鉴于目前还没有人将此方法用于膜蛋白的结构预测.我们拟进一步发展此方法.     

Functional expression of green fluorescent protein derivatives in Halobacterium salinarum

We investigated the applicability of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for gene expression in an extremely halophilic organism: Halobacterium salinarum. Two recombinant GFPs were fused with bacteriorhodopsin, a typical membrane protein of H. salinarum. These fusion proteins preserved the intrinsic functions of each component, bacteriorhodopsin and GFP, were expressed in H. salinarum under conditions with an extremely high salt concentration, and were proved to be properly localized in its plasma membrane. These results suggest that GFP could be used as a versatile reporter of gene expression in H. salinarum for investigations of various halophilic membrane proteins, such as sensory rhodopsin or phoborhodopsin.     

A new approach to understanding the initial step in visual transduction.

Data from picosecond spectroscopic studies of the formation kinetics of bathorhodopsin upon photolysis of rhodopsin and isorhodopsin was analyzed in terms of the Englman-Jortner theory of radiationless transitions. It was found that low frequency vibrations of the protein and/or chromophore are important in coupling bathorhodopsin to its precursor. The results were consistent with a mechanism for bathorhodopsin formation involving only a simple chromophore isomerization. A similar analysis of the formation kinetics of the K state of bacteriorhodopsin showed that different low frequency vibrations than those calculated for rhodopsin couple it to its precursor. The frequency of these vibrations increases upon deuteration for rhodopsin, while it decreases upon deuteration for bacteriorhodopsin. This points out the importance the specific protein matrix has on the primary photolysis reaction.     

Solid-state 2H NMR spectroscopy of retinal proteins in aligned membranes

Solid-state 2H 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. 2H 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-C2H3 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 2H 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 beta-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H 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 2H NMR is thus illuminating in terms of their different biological functions.     

Precision of sodium dodecyl sulfate-polyacrylamide-gel electrophoresis for the molecular weight estimation of a membrane glycoprotein: studies on bovine rhodopsin.

Criteria for assessing the precision and accuracy of methods for estimation of molecular weight for proteins using sodium dodecyl sulfate-polyacrylamide-gel electrophoresis have been applied to rhodopsin from bovine visual cell outer segment membranes. Various methods of preparing this hydrophobic protein for electrophoresis differ in their ability to solubilize and disaggregate polypeptide constituents of the outer segment membrane, with resultant variations in the pattern of protein bands and the apparent molecular weight of rhodopsin. Even with optimal solubilization and disaggregation, the behavior of rhodopsin relative to a series of standard proteins is such that the apparent molecular weight decreases systematically from 40,400 to 34,500 as the acrylamide concentration increases from 4 to 10%. As demonstrated by Ferguson plots of logR_f vs gel concentration and split gel experiments, this discrepancy is explained by the fact that the extrapolated R_f for zero gel concentration (Y₀) for rhodopsin is significantly lower than the Y₀'s for the soluble proteins used as molecular weight standards. In such cases, a possibly more reliable molecular weight estimate is obtained by plotting the retardation coefficient (K_R) vs molecular weight. This method yields a value of 29,500 ± 1000 for bovine rhodopsin if only the errors in measurement of R_f are considered and a quadratic relationship between K_R and molecular weight is used. Using weighted linear regression for K_R vs molecular weight, we obtain a molecular weight estimate of 32,700 ± 5000 when the uncertainty in the calibration curve is considered. Because of uncertainties regarding the detergent-binding properties of rhodopsin and the relationship of its Stokes radius to its molecular weight by comparison with the soluble protein standards, these values must be viewed with caution.     

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.     

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.