The secondary structure of bacteriorhodopsin determined by Raman and circular dichroism spectroscopy

The secondary structure of bacterio-opsin (BO), the retinal free protein-component of bacteriorhodopsin (BR), has been determined by Raman spectroscopy. Additional circular dichroism (CD) measurements have revealed only negligible conformational differences between BO in apomembranes and BR in purple membranes. Therefore, the secondary structure of BR was derived from the Raman data of BO. The protein conformation was determined to consist of 72-82% helices, 2-11% beta-strands, and 11-17% beta-turns. Only about half of the helical structures correspond to alpha 1-helices, the other half possess non-alpha 1-helical structures. According to the analysis of the Raman data, the derived secondary structure of BR was obtained with high reliability for all structure classes which can be distinguished by this method within the given uncertainty range. This is a remarkable difference from recently published secondary structural data derived from CD measurements where the helix content was reported to be between 50 and 80%. The inherent experimental and methodological uncertainties of the CD-technique leading to such a range of variation are critically discussed in comparison to the method of Raman spectroscopy. The combined application of Raman and CD spectroscopy, as performed here, is demonstrated to be a substantial improvement in the secondary structure determination of retinal-containing membrane proteins. On the basis of our results, some of the recently proposed structural models of BR with a beta-strand content of more than 11% can be ruled out.     

Circular dichroism and cross-linking studies of bacteriorhodopsin mutants

Summary. Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.     

Orientation of the protonated retinal Schiff base group in bacteriorhodopsin from absorption linear dichroism.

Linear dichroism experiments are performed on light-adapted bacteriorhodopsin (BR568) films containing native retinal (A1) and its 3,4-dehydroretinal (A2) analogue to measure the angle between the chromophore transition dipole moment and the membrane normal. QCFF/pi calculations show that the angle between the transition moment and the long axis of the polyene is changed by 3.4 degrees when the C3-C4 bond is unsaturated. The difference vector between the two transition moments points in the same direction as the Schiff base (N----H) bond for the all-trans BR568 chromophore. Because the plane of the chromophore is perpendicular to the membrane plane, a comparison of the transition moment orientations in the A1- and A2-pigments enables us to determine the orientation of the N----H bond with respect to the absolute chromophore (N----C5 vector) orientation. The angles of the transition moments are 70.3 degrees +/- 0.4 degrees and 67.8 degrees +/- 0.4 degrees for the A1- and A2-pigments, respectively. The fact that the change in the transition moment angle (2.5 degrees) is close to the predicted 3.4 degrees supports the idea that the chromophore plane is nearly perpendicular to the membrane plane. The decreased transition moment angle in the A2-analogue requires that the N----H bond and the N----C5 vector point toward the same membrane surface. Available results indicate that the N----C5 vector points toward the exterior in BR568. With this assignment, we conclude that the N----H bond points toward the exterior surface and its most likely counterion Asp-212. This information makes possible the construction of a computer graphics model for the active site in BR568.     

X-ray diffraction of bacteriorhodopsin photocycle intermediates

Recent advances in the crystallography of bacteriorhodopsin, the light-driven proton pump, have yielded structural models for all intermediates of the photochemical cycle. For seven of the species, X-ray diffraction data were collected from trapped photostationary states in crystals, and for the two remaining ones the structures of selected mutants are available. The changes of the retinal chromophore, protein and bound water describe, at an atomic level, how accommodation of the twisted photoisomerized retinal to its binding site causes de-protonation of the retinal Schiff base and initiates cascades of gradual conformational rearrangements of the protein. One cascade propagates in the extracellular direction and results in proton release, and the other in the cytoplasmic direction and results in side-chain and main-chain rearrangements, formation of a chain of hydrogen-bonded water, and proton uptake from the bulk. Such local-global conformational coupling, with gradual spreading of a local perturbation over the rest of the protein, might be the uniting principle of transporters and receptors.     

Protein secondary structure from circular dichroism spectra

Circular dichroism spectra of proteins are extremely sensitive to secondary structure. Nevertheless, circular dichroism spectra should not be analyzed for protein secondary structure unless they are measured to at least 184 nm. Even if all the various types ofβ-turns are lumped together, there are at least 5 different types of secondary structure in a protein (α-helix, antiparallelβ-sheet, parallelβ-sheet,β-turn, and other structures not included in the first 4 categories). It is not possible to solve for these 5 parameters unless there are 5 equations. Singular value decomposition can be used to show that circular dichroism spectra of proteins measured to 200 nm contain only 2 pieces of information, while spectra measured to 190 nm contain about 4. Adding the constraint that the sum of secondary structures must equal 1 provides another piece of information, but even with this constraint, spectra measured to 190 nm simply do not analyze well for the 5 unknowns in secondary structure. Spectra measured to 184 nm do contain 5 pieces of information and we have used such spectra successfully to analyze a variety of proteins for their component secondary structures.     

Infrared dichroism of isotope-edited alpha-helices and beta-sheets

Isotope editing of amide infrared bands not only localises secondary structural elements within the protein but also yields conformational information that is not available from the linear dichroism of aligned samples without isotope editing. The additional information that can be derived on the orientational distribution of alpha-helices in membranes by the combined use of different amide bands and several positions of labelling is presented here. Also, the relationship between the azimuthal orientation of the transition moment and the protein structure is treated explicitly. A comprehensive analysis of the infrared dichroism for beta-sheets and beta-barrels is given here, for the first time. The orientation of the individual transition moments in a beta-sheet that is essential for this analysis is derived for the different amide bands.     

Circular dichroism of halorhodopsin: comparison with bacteriorhodopsin and sensory rhodopsin I

Circular dichroic (CD) spectra of three related protein pigments from Halobacterium halobium, halorhodopsin (HR), bacteriorhodopsin (BR), and sensory rhodopsin I (SR-I), are compared. In native membranes the two light-driven ion pumps, HR and BR, exhibit bilobe circular dichroism spectra characteristic of exciton splitting in the region of retinal absorption, while the phototaxis receptor, SR-I, exhibits a single positive band centered at the SR-I absorbance maximum. This indicates specific aggregation of protein monomers of HR, as previously noted [Sugiyama, Y., & Mukohata, Y. (1984) J. Biochem. (Tokyo) 96, 413-420], similar to the well-characterized retinal/retinal exciton interaction in the purple membrane. The absence of this interaction in SR-I indicates SR-I is present in the native membrane as monomers or that interactions between the retinal chromophores are weak due to chromophore orientation or separation. Solubilization of HR and BR with nondenaturing detergents eliminates the exciton coupling, and the resulting CD spectra share similar features in all spectral regions from 250 to 700 nm. Schiff-base deprotonation of both BR and HR yields positive CD bands near 410 nm and shows similar fine structure in both pigments. Removal of detergent restores the HR native spectrum. HR differs from BR in that circular dichroic bands corresponding to both amino acid and retinal environments are much more sensitive to external salt concentration and pH. A theoretical analysis of the exciton spectra of HR and BR that provides a range of interchromophore distances and orientations is performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallographic characterization by X-ray diffraction of the M-intermediate from the photo-cycle of bacteriorhodopsin at room temperature

The structure of the M-intermediate appearing in the photo-cycle of bacteriorhodopsin was studied with X-ray diffraction techniques at room temperature. The lifetime of the M-intermediate was prolonged by treatment with an arginine solution at alkaline pH (Nakasako et al., FEBS Lett. 254, 211-214). The diffraction profile of membranes which had accumulated the M-intermediate had small but significant differences in the intensities of Bragg reflections and the lattice constant in comparison with that of membranes having trans-bacteriorhodopsin. Diffraction intensities were carefully evaluated and the structural changes during the formation of the intermediate were evaluated with difference Fourier analysis. We could find structural changes around helices G and B.     

X-ray-radiation-induced changes in bacteriorhodopsin structure

Bacteriorhodopsin (bR) provides light-driven vectorial proton transport across a cell membrane. Creation of electrochemical potential at the membrane is a universal step in energy transformation in a cell. Published atomic crystallographic models of early intermediate states of bR show a significant difference between them, and conclusions about pumping mechanisms have been contradictory. Here, we present a quantitative high-resolution crystallographic study of conformational changes in bR induced by X-ray absorption. It is shown that X-ray doses that are usually accumulated during data collection for intermediate-state studies are sufficient to significantly alter the structure of the protein. X-ray-induced changes occur primarily in the active site of bR. Structural modeling showed that X-ray absorption triggers retinal isomerization accompanied by the disappearance of electron densities corresponding to the water molecule W402 bound to the Schiff base. It is demonstrated that these and other X-ray-induced changes may mimic functional conformational changes of bR leading to misinterpretation of the earlier obtained X-ray crystallographic structures of photointermediates.     

Theoretical modeling of the O-intermediate structure of bacteriorhodopsin

Bacteriorhodopsin is a prototype of efficient molecular machinery functioning as a light-activated proton pump. Among the five distinct intermediates (K, L, M, N, and O) of the photocycle, there is less structural information on the later stages compared with the early intermediates. Here, we report the structural modeling of the O-intermediate for which the determination of experimental structure remains difficult. Hypothetical conformational change of the molecule from the light-adapted state to the O-intermediate state was simulated by gradually changing the protonation state of two residues. To achieve accurate molecular modeling, we carefully constructed a realistic system of the native purple membrane. The modeled structure of the O-intermediate has some implications about proton transfer in the later stages of the photocycle and the structural response of bacteriorhodopsin to the inner charge distribution.     

Infrared studies of water induced conformational changes in bacteriorhodopsin

Fourier transform infrared difference spectroscopy has been used to study the effect of water on the conformation of bacteriorhodopsin. The infrared spectra as a function of water content show a conformational change at about 0.06 g H₂O/g bacteriorhodopsin. By an interference method the thickness of the sample was measured and shows similar behavior as a function of water content. This study gives insight into the process of water absorption by purple membrane. The observations are in good agreement with those found for other proteins.Abbreviations IR infrared - FTIR Fourier transform IR     

Cavity depth on the bacteriorhodopsin peptide surface near the proton carrier Asp96: Data of X-ray structure models

Three-dimensional X-ray models of the wild-type bacteriorhodopsin structure are investigated by means of the program PyMOL. Construction of the surfaces accessible to the solvent at the cytoplasmic side visualized a cavity near the proton carrier Asp96. The cavity shortens the way of the proton from the membrane surface to this carrier. The distance between the cavity surface and the centre of the carbonic atom of the Asp96 carboxylic group is ～6 Å. Besides, for model structures 1c3w, 1qhj, and 1BRR, a channel of radius 1.1 Å is revealed between the cytoplasmic surface and Asp96carboxyl. The channel diameter is narrower than the characteristic diameter of the water molecule and apparently does not create conductivity in the nonexcited pigment. It is possible however that along this channel a hydrated “gap” opens at the second phase of a bacteriorhodopsin photocycle related with reprotonation of Asp96.     

Peptide-chain secondary structure of bacteriorhodopsin.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.     

Infrared dichroism of twisted beta-sheet barrels. The structure of E. coli outer membrane proteins

The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis. For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet. Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels. Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees. Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel.     

Solution structure of the loops of bacteriorhodopsin closely resembles the crystal structure

Bacteriorhodopsin is one of very few transmembrane proteins for which high resolution structures have been solved. The structure shows a bundle of seven helices connected by six turns. Some turns in proteins are stabilized by short range interactions and can behave as small domains. These observations suggest that peptides containing the sequence of the turns in a membrane protein such as bacteriorhodopsin may form stable turn structures in solution. To test this hypothesis, we determined the solution structure of three peptides each containing the sequence of one of the turns in bacteriorhodopsin. The solution structures of the peptides closely resemble the structures of the corresponding turns in the high resolution structures of the intact protein.     

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.     

The circular dichroism and X-ray diffraction of DNA condensed from ethanolic solutions.

It is known that DNA in aqueous-ethanol solutions undergoes a B to A conformational change between 60% and 80% (w/w) ethanol. We have found that precipitates formed by adding salt to DNA in 60% and 80% ethanolic solutions can be very different. DNA precipitated from 60% ethanol forms a fine condensate that only slowly settles out of suspension and shows a characteristic differential scattering of circularly polarized light at long wavelengths. DNA precipitated from 80% ethanol forms a flocculent aggregate that exhibits the CD spectral features of the A conformation. Data from circular dichroism spectra of natural and synthetic nucleic acids and from X-ray diffraction patterns of the precipitates show that DNA molecules precipitated from 60% and 80% ethanol are, respectively, in the B and A conformation. Therefore, the different secondary conformations of DNA in ethanolic solutions are maintained during precipitation under these conditions. These results are of general importance for the preparation and study of condensed forms of DNA, since a relatively small change in the extent of dehydration can change the secondary conformation of DNA and markedly affect the character of a subsequent precipitate.