NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)     

FTIR spectroscopy of the K photointermediate of Neurospora rhodopsin: structural changes of the retinal, protein, and water molecules after photoisomerization

Neurospora rhodopsin (NR, also known as NOP-1) is the first rhodopsin of the haloarchaeal type found in eucaryotes. NR demonstrates a very high degree of conservation of the amino acids that constitute the proton-conducting pathway in bacteriorhodopsin (BR), a light-driven proton pump of archaea. Nevertheless, NR does not appear to pump protons, suggesting the absence of the reprotonation switch that is necessary for the active transport. The photocycle of NR is much slower than that of BR, similar to the case of pharaonis phoborhodopsin (ppR), an archaeal photosensory protein. The functional and photochemical differences between NR and BR should be explained in the structural context. In this paper, we studied the structural changes of NR following retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy and compared the obtained spectra with those for BR. For the spectroscopic analysis, we established the light-adaptation procedure for NR reconstituted into 1,2-dimyristoyl-sn-glycero- 3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA) liposomes, which takes approximately 2 orders of magnitudes longer than in BR. The structure of the retinal chromophore and the hydrogen-bonding strength of the Schiff base in NR are similar to those in BR. Unique spectral features are observed for the S-H stretching vibrations of cysteine and amide-I vibrations for NR before and after retinal isomerization. In NR, there are no spectral changes assignable to the amide bands of alpha helices. The most prominent difference between NR and BR was seen for the water O-D stretching vibrations (measured in D(2)O). Unlike for haloarchaeal rhodopsins such as BR and ppR, no O-D stretches of water under strong hydrogen-bonded conditions (<2400 cm(-1)) were observed in the NR(K) minus NR difference spectra. This suggests a unique hydrogen-bonded network of the Schiff base region, which may be responsible for the lack of the reprotonation switch in NR.     

Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction

Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussed in the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.     

Reformation of crystalline purple membrane from purified bacteriorhodopsin fragments.

Reconstituted crystalline purple membrane has been prepared starting from denatured bacteriorhodopsin (BR) fragments, native lipids and retinal. The two chymotryptic fragments are thought to contain respectively five and two transmembrane alpha-helices in native BR. The new reconstitution procedure, a modification of that of Huang et al. (1986, J. Biol. Chem., 256, 3802), relies on dodecylsulfate precipitation by potassium ions and yields samples with a high protein-to-lipid ratio (approximately 1:1 w/w). X-ray and neutron diffraction measurements show that in the reconstituted samples BR molecules are arranged in a P3 two-dimensional lattice with the same unit cell dimensions as the native purple membrane lattice. Analysis of reflection intensities indicates that the reconstituted molecules have regained the structure of native BR to 7 A resolution.     

Localization of two chymotryptic fragments in the structure of renatured bacteriorhodopsin by neutron diffraction.

The structure of crystalline purple membrane reconstituted from purified bacteriorhodopsin (BR) chymotryptic fragments has been studied by neutron diffraction. In one of the samples studied, the fragment C-2, encompassing the first two predicted transmembrane segments, was prepared from deuterated purple membrane. The diffraction changes when the natural C-2 fragment is substituted by a deuterated one are analysed in terms of a seven-helix model for BR. The assignment of the labelled fragment to one end of the molecule placed new constraints on folding models for the protein.     

Packing of transmembrane helices in bacteriorhodopsin folding: structure and thermodynamics

We propose a coarse-grained (CG) model to study the native structure and physical properties of helical membrane proteins (HMPs) using off-lattice computer simulations. Instead of considering sequence heterogeneity explicitly, we model its effect on the packing of helices by employing a mean packing parameter r(0), which is calculated from an all-atom (AA) model. Specifically, this CG model is applied to investigate the packing of helices in bacteriorhodopsin (BR), and predicts the seven helix bundle structure of BR with a root mean square deviation (RMSD) in coordinates of helix backbone atoms (N, C, C(alpha)) of 3.99 A from its crystal structure. This predicted structure is further refined in an AA model by Amber and the refined structure has a RMSD (in coordinates of helix backbone atoms) of 2.64 A. The predicted packing position, tilting angle, and orientation angle of each helix in the refined structure are consistent with experimental data and their physical origins can be well understood in our model. Our results show that a reasonably good structure of BR can be predicted by using such a dual-scale approach, provided that its secondary structure is known. Starting from a random initial configuration, the folded structure can be obtained in days using a regular desktop computer. Various thermodynamic properties of helix packing of BR are also investigated in this CG model.     

Identification of Helical Packing Motifs Common to Bacteriorhodopsin and G Protein-Coupled Receptors

A sequence analysis and comparison of transmembrane helices in bacteriorhodopsin (BR) and G protein-coupled receptors (GPCRs) is presented to identify potential regions of homology across protein families. The results show a common pattern of residues is conserved within the interhelical contact regions of BR that fit a knob-into-hole structural motif previously postulated for globular proteins and photosynthetic reaction centers. Based on an alignment of conserved prolines in transmembrane helices, it is inferred that analogous helix packing arrangements are possible in the rhodopsin-like GPCRs. Molecular models of GPCR helices V and VI indicate these interactions occur between aromatic and hydrophobic residues flanking the highly conserved prolines in these sequences. A similar packing arrangement is shown to occur in the X-ray structure of the melittin which also displays a unique pairing of proline-linked helices. The contact pattern identified is further applied to predict the packing of pairs of proline-containing helices in the pheromone-like and cAMP GPCRs. A potential role in stabilizing structure formation is also suggested for the contacts. The results and conclusions are supported by recent biophysical studies of zinc binding to kappa-opioid receptor mutants.     

Identification of Helical Packing Motifs Common to Bacteriorhodopsin and G Protein-Coupled Receptors

A sequence analysis and comparison of transmembrane helices in bacteriorhodopsin (BR) and G protein-coupled receptors (GPCRs) is presented to identify potential regions of homology across protein families. The results show a common pattern of residues is conserved within the interhelical contact regions of BR that fit a knob-into-hole structural motif previously postulated for globular proteins and photosynthetic reaction centers. Based on an alignment of conserved prolines in transmembrane helices, it is inferred that analogous helix packing arrangements are possible in the rhodopsin-like GPCRs. Molecular models of GPCR helices V and VI indicate these interactions occur between aromatic and hydrophobic residues flanking the highly conserved prolines in these sequences. A similar packing arrangement is shown to occur in the X-ray structure of the melittin which also displays a unique pairing of proline-linked helices. The contact pattern identified is further applied to predict the packing of pairs of proline-containing helices in the pheromone-like and cAMP GPCRs. A potential role in stabilizing structure formation is also suggested for the contacts. The results and conclusions are supported by recent biophysical studies of zinc binding to kappa-opioid receptor mutants.     

Thermal denaturing of bacteriorhodopsin by X-Ray scattering from oriented purple membranes

We present a temperature-dependent x-ray diffraction study of thin films of purple membranes (PMs) with the native membrane protein bacteriorhodopsin (BR). The high degree of alignment with respect to the silicon substrates allows for the application of modern interface-sensitive scattering techniques. Here we focus on the structural changes of BR in PMs at the thermal denaturing transition. A partial unfolding of the helices is observed rather than the complete unfolding process known from helix to coil transitions. While BR remains threaded into the lipid bilayer in the denatured state, changes in the short-range lateral structures are associated with the partial unfolding of the transmembrane helices.     

Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.     

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.     

Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.     

Self-association of transmembrane alpha-helices in model membranes: importance of helix orientation and role of hydrophobic mismatch

Interactions between transmembrane helices play a key role in almost all cellular processes involving membrane proteins. We have investigated helix-helix interactions in lipid bilayers with synthetic tryptophan-flanked peptides that mimic the membrane spanning parts of membrane proteins. The peptides were functionalized with pyrene to allow the self-association of the helices to be monitored by pyrene fluorescence and Trp-pyrene fluorescence resonance energy transfer (FRET). Specific labeling of peptides at either their N or C terminus has shown that helix-helix association occurs almost exclusively between antiparallel helices. Furthermore, computer modeling suggested that antiparallel association arises primarily from the electrostatic interactions between alpha-helix backbone atoms. We propose that such interactions may provide a force for the preferentially antiparallel association of helices in polytopic membrane proteins. Helix-helix association was also found to depend on the lipid environment. In bilayers of dioleoylphosphatidylcholine, in which the hydrophobic length of the peptides approximately matched the bilayer thickness, association between the helices was found to require peptide/lipid ratios exceeding 1/25. Self-association of the helices was promoted by either increasing or decreasing the bilayer thickness, and by adding cholesterol. These results indicate that helix-helix association in membrane proteins can be promoted by unfavorable protein-lipid interactions.     

Topological arrangement of the intracellular membrane fusion machinery

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form a four-helix coiled-coil bundle that juxtaposes two bilayers and drives a basal level of membrane fusion. The Sec1/Munc18 (SM) protein binds to its cognate SNARE bundle and accelerates the basal fusion reaction. The question of how the topological arrangement of the SNARE helices affects the reactivity of the fusion proteins remains unanswered. Here we address the problem for the first time in a reconstituted system containing both SNAREs and SM proteins. We find that to be fusogenic a SNARE topology must support both basal fusion and SM stimulation. Certain topological combinations of exocytic SNAREs result in basal fusion but cannot support SM stimulation, whereas other topologies support SM stimulation without inducing basal fusion. It is striking that of all the possible topological combinations of exocytic SNARE helices, only one induces efficient fusion. Our results suggest that the intracellular membrane fusion complex is designed to fuse bilayers according to one genetically programmed topology.     

Properties of bacteriorhodopsin derivatives constructed by insertion of an exogenous epitope into extra-membrane loops.

Bacteriorhodopsin (BR) is folded into a bundle of seven alpha-helices which is embedded in the cellular membrane of Halobacterium salinarium; these helices are connected by short extra-membrane loops, three on the cytoplasmic side and three on the outside. Oligonucleotide-directed insertion or replacement mutagenesis was used to integrate the C-terminal sequence (13 amino acids long) of Sendai virus L-protein individually into each of the six helix-connecting loops. The altered gene products were obtained by expression of the mutant genes in either Escherichia coli or Schizosaccharomyces pombe and were used to reconstitute BR in proteoliposomes. In four cases (altered loops B/C, C/D, D/E or E/F), the mutant BRs were found to be fully functional as judged by light-driven proton pumping and photocycle kinetics. Within the four functional BR variants, recognition of the viral epitope by a monoclonal antibody is restricted to modified loops B/C and E/F. Immunogold staining of S.pombe cells producing either of the two latter BR variants shows that the protein is distributed among various cellular membranes but is not present in mitochondrial membranes. Sequence alteration of loop A/B or F/G resulted in loss of function, most plausibly due to a folding defect of the respective proteins. These results on the one hand document differences in structural importance of the various BR extra-membrane loops and on the other hand open the door to the construction of multifunctional membrane proteins via loop replacement mutagenesis of BR.     

Thermodynamic measurements of the contributions of helix-connecting loops and of retinal to the stability of bacteriorhodopsin.

Thermodynamic studies of bacteriorhodopsin (BR) have been undertaken in order to investigate the factors that stabilize the structure of a membrane protein. The stability of the native, intact protein was compared to that of protein with retinal removed, and/or cleaved in one or two of the loops connecting the transmembrane helices. The stability was assessed using differential scanning calorimetry and thermal denaturation curves obtained from ultraviolet circular dichroism and absorption spectroscopy. Retinal binding and the loop connections were each found to make a small contribution to stability, and even a sample that was cleaved twice as well as bleached to remove retinal denatured well above room temperature. Removal of retinal destabilized the protein more than cleaving once, and about as much as cleaving twice. Retinal binding and the connections in the loops were found to stabilize BR in independent ways. Cleavage of the molecule into fragments did not reduce the intermolecular cooperativity of the denaturation. Dilution of the protein by addition of excess lipid in order to eliminate the purple membrane crystal lattice also did not alter the cooperativity. These results are used to compare the relative importance of various contributors to the stability of BR.     

Modeling of halorhodopsin and rhodopsin based on bacteriorhodopsin

Bacteriorhodopsin (BR), halorhodopsin (HR), and rhodopsin (Rh) all belong to the class of seven-helix membrane proteins. For BR, a structural model at atomic resolution is available; for HR, diffraction data are available only down to a resolution of 6 Å in the membrane plane, and for Rh, down to 9 Å. BR and HR are closely related proteins with a sequence homology of 34%, while Rh does not share any sequence homology with BR. An atomic model for HR is derived that is based on sequence alignment and the atomic model for BR and is improved by molecular dynamics simulations. The model structure obtained accounts well for the experimentally observed difference between HR and BR in the projection map, where HR exhibits a higher density in the region between helices D and E. The reason for this difference lies partially in the different side chains and partially in slightly different helix tilts. The scattering amplitudes and phases of the model structure are calculated and agree with the experimental data down to a resolution of about 8 Å. If the helix positions are adopted from the projection map for HR and used as input in the model, this number improves to 7 Å. Analogously, an atomic model for Rh is derived based on the atomic model for BR and subjected to molecular dynamics simulations. Optimal agreement with the experimental projection map for Rh is obtained when the entire model structure is rotated slightly about two axes in the membrane plane. The agreement with the experimental projection map is not as satisfactory as for HR, but the results indicate that even for a nonhomologous, but structurally related, protein such as Rh, an acceptable model structure can be derived from the structure of BR. © 1996 Wiley-Liss, Inc.     

Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein associated with high density lipoprotein (HDL), and its plasma levels have been correlated with protection against atherosclerosis. Unfortunately, the structural basis of this phenomenon is not fully understood. Over 25 years of study have produced two general models of apoA-I structure in discoidal HDL complexes. The "belt" model states that the amphipathic helices of apoA-I are aligned perpendicular to the acyl chains of the lipid bilayer, whereas the "picket fence" model argues that the helices are aligned parallel with the acyl chains. To distinguish between the two models, various single tryptophan mutants of apoA-I were analyzed in reconstituted, discoidal HDL particles composed of phospholipids containing nitroxide spin labels at various positions along the acyl chain. We have previously used this technique to show that the orientation of helix 4 of apoA-I is most consistent with the belt model. In this study, we performed additional control experiments on helix 4, and we extended the results by performing the same analysis on the remaining 22-mer helices (helices 1, 2, 5, 6, 7, 8, and 10) of human apoA-I. For each helix, two different mutants were produced that each contained a probe Trp occurring two helical turns apart. In the belt model, the two Trp residues in each helix should exhibit maximal quenching at the same nitroxide group position on the lipid acyl chains. For the picket fence model, maximal quenching should occur at two different levels in the bilayer. The results show that the majority of the helices are in an orientation that is consistent with a belt model, because most Trp residues localized to a position about 5 A from the center of the bilayer. This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs.     

A hydrogen bonded chain in bacteriorhodopsin by computer modelling approach

The seven alpha-helical segments of Bacteriorhodopsin (BR) passing through the membrane are investigated for a continuous Hydrogen Bonded Chain (HBC). The study is carried out by computer modelling approach. It is assumed that the seven helices are placed as (AGFEDCB), which has been accepted as the best model by several groups. Helices A, D, E and G are considered to be present in right handed alpha-helical conformation. The inter-orientation of these helices are represented by Eulerian angles alpha, beta and gamma. For the helices B, C and F which contain Proline in the middle, several conformational possibilities were considered. In these cases apart from the Eulerian angles alpha, beta and gamma, the dihedral angles phi p-1 and psi p-1 of the residues that are succeeded by Proline residue in the helical regions were also used in fixing the position of the helices with respect to each other. All these parameters were varied to fit with the top, middle and bottom distances reported by electron diffraction studies. Good fit was obtained for all right handed alpha-helical conformations and also for helices B, C and F with a left handed turn at the residue preceeding proline. Hence two structures were analysed for continuous HBC. Structure I which contained all the seven helices in right handed alpha-helical conformation and Structure II, which had the helices A, D, E and G in right handed conformation and the helices B, C and F in right handed alpha-helical conformation with a left handed turn at the residue preceeding proline. All possible staggered conformations were considered for the side chains and the inter atomic distances were analysed for Hydrogen bonds. It was possible to obtain a continuous chain in both the structures which includes most of the residues found to be important by the experiments. However Lys-216 has to be considered in two different conformations to connect the cytoplasmic side with the extra cellular side. The overall height spanned by HBC is about 25A. The chains obtained by both the structures I and II are analysed in terms of the conformational parameters. It has also been possible to place the retinal in the region as predicted by the experiments. The Tryptophan residues which affect the spectral characteristics can be aligned on either side of the retinal.     

Protein-bound water as the determinant of asymmetric functional conversion between light-driven proton and chloride pumps

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR. Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.