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.     

Moist and soft, dry and stiff: a review of neutron experiments on hydration-dynamics-activity relations in the purple membrane of Halobacterium salinarum

Twenty-five years of neutron experiments on hydration and thermal dynamics in purple membranes of Halobacterium salinarum are reviewed. Neutron diffraction, elastic and quasielastic scattering, allowed to map the distribution of water and lipids and to measure thermal fluctuations and correlation times in the membranes, under various conditions of temperature, hydration and lipid environment. Strong correlations were established between dynamics parameters and the activity of bacteriorhodopsin (the purple membrane protein), as a light driven proton pump supporting the hypothesis that the influence of hydration on activity is in fact due to its effects on membrane thermal dynamics. Hydrogen-deuterium labelling experiments highlighted stiffer and softer parts in the bacteriorhodopsin structure. The soft parts would allow the conformational changes involved in activity, while the stiffer ones may control a valve-like function in vectorial proton transfer.     

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis

Summary Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated³H leucine from the digested membranes.During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.     

Location of Chemically Modified Lysine 41 in the Structure of Bacteriorhodopsin by Neutron Diffraction

Purple membranes were prepared in which bacteriorhodopsin was labeled at lysine 41 with phenylisothiocyanate (PITC) and with perdeuterated PITC. The in-plane position of this small label containing only five deuterons was determined from the differences between the neutron diffraction intensities of the two samples. At 8.7-Å resolution the Fourier difference map revealed a well-defined site between helices 3 and 4. This position was confirmed by a refinement procedure in reciprocal space. Model calculations showed that the observed difference density had the right amplitude for the label. Thus it is possible to locate a small group in a large protein structure by replacing as few as five hydrogens by deuterium. The observed location of PITC restricts the number of possibilities for the assignment of helix B in the sequence (to which lysine 41 is attached) to one of the seven helices of the structure. Taking into account the size of the label and the length of the lysine side chain our result excludes helices 1, 2, and 7 as candidates for B.     

Determination of the number of water molecules in the proton pathway of bacteriorhodopsin using neutron diffraction data

It has been shown that water molecules participate in the proton pathway of bacteriorhodopsin. Large efforts have been made to determine with various biophysical methods the number of water molecules involved. Neutron diffraction H2O/D2O exchange experiments have been often used to reveal the position of water even with low-resolution diffraction data. With this technique, care must be taken with the limitations of the difference Fourier method which are commonly applied to analyze the data. In this paper we compare the results of the difference Fourier method applied to measured diffraction data (not presented here) and models with those from alternative methods introduced here: (1) a computer model calculation procedure to determine a label's scattering length density based on a comparison of intensity differences derived from models and intensity differences from our measurements; (2) a method based on the Parseval formula. Both alternative methods have been evaluated and tested using results of neutron diffraction experiments on purple membranes (Hauss et al. 1994). Our findings indicate that the difference Fourier method applied to low-resolution diffraction data can successfully determine the position of localized water molecules but underestimates their integrated scattering length density in the presence of labels in other positions. Furthermore, we present the results of neutron diffraction experiments on purple membranes performed to determine the number of water molecules in the projected area of the Schiff base at 86%, 75% and 57% relative humidity (r.h.). We found 19 +/- 2 exchangeable protons at 75% r.h., which means at least 8-9 water molecules are indispensable for normal pump function.     

Charting the surfaces of the purple membrane

The preponderance of structural data of the purple membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transmembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the purple membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.     

Three-dimensional fold of the human AQP1 water channel determined at 4 A resolution by electron crystallography of two-dimensional crystals embedded in ice

Here, we present a three-dimensional (3D) density map of deglycosylated, human erythrocyte aquaporin 1 (AQP1) determined at 4 A resolution in plane and approximately 7 A resolution perpendicular to the bilayer. The map was calculated by analyzing images and electron diffraction patterns recorded from tilted (up to 60 degrees ), ice-embedded, frozen-hydrated 2D crystals of AQP1 in lipid bilayer membranes. This map significantly extends the findings related to the folding of the AQP1 polypeptide chain determined by us at a lower, 7 A by approximately 20 A, resolution. The solvent-accessible volume within a monomer has a vestibular architecture, with a narrow, approximately 6.5 A diameter constriction near the center of the bilayer, where the location of the water-selective channel is postulated to exist. The clearly resolved densities for the transmembrane helices display the protrusions expected for bulky side-chains. The density in the interior of the helix barrel (putative NPA box region) is better resolved compared to our previous map, suggesting clearer linkage to some of the helices, and it may harbor short stretches of alpha-helix. At the bilayer extremities, densities for some of the inter-helix hydrophilic loops are visible. Consistent with these observed inter-helix connections, possible models for the threading of the AQP1 polypeptide chain are presented. A preferred model is deduced that agrees with the putative locations of a group of aromatic residues in the amino acid sequence and in the 3D density map.     

Identifying anisotropic constraints in multiply labeled bacteriorhodopsin by 15N MAOSS NMR: a general approach to structural studies of membrane proteins

Structural models of membrane proteins can be refined with sets of multiple orientation constraints derived from structural NMR studies of specifically labeled amino acids. The magic angle oriented sample spinning (MAOSS) NMR approach was used to determine a set of orientational constraints in bacteriorhodopsin (bR) in the purple membrane (PM). This method combines the benefits of magic angle spinning (MAS), i.e., improved sensitivity and resolution, with the ability to measure the orientation of anisotropic interactions, which provide important structural information. The nine methionine residues in bacteriorhodopsin were isotopically (15)N labeled and spectra simplified by deuterium exchange before cross-polarization magic angle spinning (CPMAS) experiments. The orientation of the principal axes of the (15)N chemical shift anisotropy (CSA) tensors was determined with respect to the membrane normal for five of six residual resonances by analysis of relative spinning sideband intensities. The applicability of this approach to large proteins embedded in a membrane environment is discussed in light of these results.     

Structural comparison of native and deoxycholate-treated purple membrane.

Lipid-depleted purple membrane prepared by extraction with deoxycholate has been compared with the native structure. X-ray and electron diffraction photographs show a reduction in cell dimension from 62.4 to 57.3 A, and a substantial change in the distribution of diffraction intensity compared with the native specimens. Low-dose electron microscopy has been used to obtain a projected density map of lipid-depleted membranes. The projected structure shows that the deoxycholate treatment removes a boundary layer of lipid, which in the native form separates adjacent trimers of bacteriorhodopsin. The map also provides an improved estimate of the molecular envelope of the protein. A plausible arrangement for the lipid molecules in both the native and the lipid-depleted membranes is proposed, but the precise positions of individual molecules cannot yet be specified.     

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.     

Structural determinants of purple membrane assembly

The purple membrane is a two-dimensional crystalline lattice formed by bacteriorhodopsin and lipid molecules in the cytoplasmic membrane of Halobacterium salinarum. High-resolution structural studies, in conjunction with detailed knowledge of the lipid composition, make the purple membrane one of the best models for elucidating the forces that are responsible for the assembly and stability of integral membrane protein complexes. In this review, recent mutational efforts to identify the structural features of bacteriorhodopsin that determine its assembly in the purple membrane are discussed in the context of structural, calorimetric and reconstitution studies. Quantitative evidence is presented that interactions between transmembrane helices of neighboring bacteriorhodopsin molecules contribute to purple membrane assembly. However, other specific interactions, particularly between bacteriorhodopsin and lipid molecules, may provide the major driving force for assembly. Elucidating the molecular basis of protein-protein and protein-lipid interactions in the purple membrane may provide insights into the formation of integral membrane protein complexes in other systems.     

Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy

The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 A. The map of the structure has a resolution of 3.5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven alpha-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.     

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.     

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.     

Structure-function studies on bacteriorhodopsin. X. Individual substitutions of arginine residues by glutamine affect chromophore formation, photocycle, and proton translocation

We have individually replaced all 7 of the arginine residues in bacteriorhodopsin by glutamine. The mutants with substitutions at positions 7, 164, 175, and 225 showed essentially the wild-type phenotype in regard to chromophore regeneration, chromophore lambda max, and proton pumping, although the mutant Arg-175----Gln showed decreased rate of chromophore regeneration. Glutamine substitutions of Arg-82, -134, and -227 affected proton pumping ability, and caused specific alterations in the bacteriorhodopsin photocycle. Finally, electrostatic interactions are proposed between Arg-82 and -227, and specific carboxylic acid residues in helices C and G, which regulate the purple to blue transition and proton transfers during the photocycle.     

The transverse location of tryptophan residues in the purple membranes of Halobacterium halobium studied by fluorescence quenching and energy transfer

Fluorescence quenching by a series of spin-labelled fatty acids is used to map the transverse disposition of tryptophan residues in bacteriorhodopsin (the sole protein in the purple membranes of Halobacterium halobium). A new method of data analysis is employed which takes into account differences in the uptake of the quenchers into the membrane. Energy transfer from tryptophan to a set of $\text{n-(9-}\text{anthroyloxy)}$ fatty acids is used as a second technique to confirm the transverse map of tryptophan residues revealed by the quenching experiments. The relative efficiencies of quenching and energy transfer obtained experimentally are compared with those predicted on the basis of current models of bacteriorhodopsin structure. Most of the tryptophan fluorescence is located near the surface of the purple membrane. When the retinal chromophore of bacteriorhodopsin is removed, tryptophan residues deep in the membrane become fluorescent. These results indicate that the deeper residues transfer their energy to retinal in the native membrane. The retinal moiety is therefore located deep within the membrane rather than at the membrane surface.     

The distribution of tetramethylammonium ions on the surface of purple membranes

The two-dimensional distribution of deuterated tetramethylammonium (TMA⁺) ions on the surface of purple membranes of Halobacterium halobium was determined by neutron diffraction. The measurements were performed on stacks of these membranes with a high concentration of TMA⁺ molecules in the water layer between the membranes. A difference Fourier analysis of samples with deuterated and undeuterated ions showed an excess of 8.5 TMA⁺ ions per elementary cell in the lipid areas compared to the protein areas. A total number of 90 ions per elementary cell in the intermembrane space was estimated from the preparation procedure. The excess in the lipid domains may result from the higher affinity of TMA⁺ ions for the lipid head groups and/or from the fact that the protein (bacteriorhodopsin) protrudes slightly out of the lipid surface.Abbreviations BR bacteriorhodopsin - TMA Tetramethylammonium     

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.     

Transient and linear dichroism studies on bacteriorhodopsin: determination of the orientation of the 568 nm all-trans retinal chromophore

The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways.In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined.In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °.No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977).The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.     

Thermodynamic studies of purple membrane

Differential dilatometric and differential scanning calorimetric measurements have been made of purple membrane with an emphasis upon the temperature range 5 degrees C less than T less than 45 degrees C. The coefficient of thermal expansion alpha is about 7 X 10(-4)/Cdeg up to 30 degrees C and decreases at higher temperatures. The specific heat increases rapidly with temperature with absolute values in the range 0.30-0.45 cal/Cdeg per g. A nearly constant alpha juxtaposed with a rapidly increasing specific heat is similar to the properties of lipid bilayers in the gel phase and alkanes in the solid phase. This behavior is explained by the concept of hindered vibrations which would now appear to apply to at least one integral membrane protein. There may also be a small broad transition centered near 20-25 degrees C that would correspond to the melting of less than 25 degrees of freedom per bacteriorhodopsin molecule and associated lipids. Using our measured apparent specific volume the average thickness of purple membrane is calculated to be 43.5 A. The specific volume of interaction of lipids and proteins is estimated, using the amino acid sequence of bacteriorhodopsin and average amino acid volumes from structural studies of other proteins, to be about 11% of the specific volume of the purple membrane lipids or 4% of the volume of the bacteriorhodopsin protein. A positive volume of interaction is consistent with lipid-protein interactions being an important determinant of the thermodynamic properties of purple membrane.