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PubMed1989

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.

Amino Acid Sequence↗
PubMed2006

Kinetics of an individual transmembrane helix during bacteriorhodopsin folding.

The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.

Bacteriorhodopsins↗
PubMed2006

The photochemical reaction cycle of retinal reconstituted bacteriorhodopsin.

The function of three types of bacteriorhodopsins was compared: the wild-type, the bleached and retinal reconstituted and retinal deficient bacteriorhodopsin after retinal addition. The apparent pK(a) of the proton acceptor group for the bleached BR and retinal deficient BR shifted toward higher pH values compared to the wild-type BR. Fitting the photocycle model to the absorption kinetic signals for all three proteins showed the existence of the same intermediates, but the time-dependent concentration of the intermediates was different. Although measurements were made at pH 7, the absorption kinetics and photoelectric signals in both retinal reconstituted samples acted as wild-type bacteriorhodopsin at significantly higher pH. Below pH 3 the retinal deficient and reconstituted sample bleached. These results suggested that the added retinal was not able to rebind in the same position in the protein as in native bacteriorhodopsin. This points out that care should be taken, when bleached bacteriorhodopsin is reconstituted with different retinal analogs.

Bacteriorhodopsins↗
PubMed1980

Effect of high pressure on the absorption spectrum and isomeric composition of bacteriorhodopsin.

The effects of high pressure upon the absorption spectra and isomeric composition of the dark (bRD) and light adapted (bRL) forms of bacteriorhodopsin were examined. Pressure favors the 13-cis form of bacteriorhodopsin (bR13-cis). The equilibrium isomeric composition and absorption spectra of bacteriorhodopsin samples at a given pressure were the same starting from either light or dark adapted bacteriorhodopsin. From the effect of pressure on the equilibrium constant between bRall-trans in equilibrium bR13-cis in the dark, the molar volume change between bRall-trans and bR13-cis was found to be -7.8 +/- 3.2 ml/mol. This volume change suggests a difference in conformation between dark- and light-adapted bacteriorhodopsin, but the magnitude of the change is small, involving only a small number of the protein residues.

Bacteriorhodopsins↗
PubMed1988

Photochemistry of monomethylated and permethylated bacteriorhodopsin.

Methylation of the nonactive site lysines of bacteriorhodopsin to form permethylated bacteriorhodopsin does not interfere with the formation of the short wavelength intermediate M412 or light-induced proton release/uptake. The absorption spectrum is similar to that of the native bacteriorhodopsin. However, additional monomethylation of the active site lysine of bacteriorhodopsin causes a red shift of the absorption maximum from 568 nm in light-adapted bacteriorhodopsin [BR] to 630 nm. The photochemistry of active-site methylated BR does not proceed beyond the L-photointermediate. In particular, the photointermediate corresponding to M412 does not form, and there is no proton pumping. Moreover, there is no tyrosine deprotonation. Thus, the formation of an M-type photointermediate is required for proton pumping by BR.

Bacteriorhodopsins↗
PubMed1996

Electrostatics and electrodynamics of bacteriorhodopsin.

The stationary electric dichroism of bacteriorhodopsin is in qualitative, but not quantitative, agreement with the orientation function for disks having a permanent dipole directed perpendicular to the plane and an induced dipole in the plane. Fits of the orientation function to data measured at low field strengths demonstrate: an increase of the permanent dipole moment mu with the square of the disk radius r2, whereas the polarizability alpha increases with r4; the ionic strength dependence is small for mu and clearly stronger for alpha; the permanent dipole moment is 4x10(6) D at r = 0.5 micron. According to the risetime constants, the induced dipole does not saturate and increases to 4x10(8) D at 40 kV/cm and r = 0.5 micron. The data indicate that the permanent dipole is not of some interfacial character but is due to a real assymetry of the charge distribution. The experimental dipole moment per protein monomer is approximately 55 D, whereas calculations based on the structure of Grigorieff et al. (Grigorieff, N., T.A. Ceska, K.H. Downing, J.M. Baldwin, and R. Henderson. 1996. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259:393-421) provide a dipole moment of approximately 570 D. The difference is probably due to a nonsymmetric distribution of charged lipid residues. It is concluded that experimental dipole moments reflect the mu-potential at the plane of shear for rotational diffusion, in analogy to the sigma-potential used for translational diffusion. It is suggested that the permanent dipole of bacteriorhodopsin supports proton transport by attraction of protons inside and repulsion of protons outside of the cell. Dichroism rise curves at field strengths between E = 150 and 800 V/cm reveal an exponential component with time constants tau 3r in the range between 1 and 40 ms, which is not found in Brownian dynamics simulations on a disk structure using hydrodynamic and electric parameters characteristic of bacteriorhodopsin disks. The experimental data suggest that this process reflects a cooperative change of the bacteriorhodopsin structure, which is induced already at a remarkably low field strength of approximately 150 V/cm.

Bacteriorhodopsins↗
PubMed1998

Photovoltage evidence that Glu-204 is the intermediate proton donor rather than the terminal proton release group in bacteriorhodopsin.

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. A two-fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of M intermediate formation in the wild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bacteriorhodopsin ground state and transition of the purple neutral form into the blue acid form. Thus, the pK of E204 in the M state is close to the pK of D85 in the bacteriorhodopsin ground state (< 3) and far below the pK of the terminal proton release group (approximately 6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.

Bacteriorhodopsins↗
PubMed1999

Membrane potential stabilizes the O intermediate in liposomes containing bacteriorhodopsin.

In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple-blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5-7 range. The efficiency of the long-lived O intermediate formation increases at a low pH. In the wild-type as well as in the E204Q mutant purple membrane, the O intermediate decay is slowed down at slightly higher pH values than that of the purple-blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the O intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H(+). At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the O intermediate. Under such conditions, membrane potential stabilization of the O intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85.

Bacteriorhodopsins↗
PubMed2003

Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.

The conformation of the AB-loop and EF-loop of bacteriorhodopsin and of the fourth cytoplasmic loop (helix VIII) of bovine rhodopsin were assessed by a combination of time-resolved fluorescence depolarization and site-directed fluorescence labeling. The fluorescence anisotropy decays were measured employing a tunable Ti:sapphire laser/microchannel plate based single-photon counting apparatus with picosecond time resolution. This method allows measurement of the diffusional dynamics of the loops directly on a nanosecond time-scale. We implemented the method to study model peptides and two-helix systems representing sequences of bacteriorhodopsin. Thus, we systematically analyzed the anisotropic behavior of four different fluorescent dyes covalently bound to a single cysteine residue on the protein surface and assigned the anisotropy decay components to the modes of motion of the protein and its segments. We have identified two mechanisms of loop conformational changes in the functionally intact proteins bacteriorhodopsin and bovine rhodopsin. First, we found a surface potential-dependent transition between two conformational states of the EF-loop of bacteriorhodopsin, detected with the fluorescent dye bound to position 160. A transition between the two conformational states at 150mM KCl and 20 degrees C requires a surface potential change that corresponds to Deltasigma approximately -1.0e(-)/bacteriorhodopsin molecule. We suggest, that the surface potential-based switch of the EF-loop is the missing link between the movement of helix F and the transient surface potential change detected during the photocycle of bacteriorhodopsin. Second, in the visual pigment rhodopsin, with the fluorescent dye bound to position 316, a particularly striking pH-dependent conformational change of the fourth loop on the cytoplasmic surface was analyzed. The loop mobility increased from pH 5 to 8. The midpoint of this transition is at pH 6.2 and correlates with the midpoint of the pH-dependent equilibrium between the active metarhodopsin II and the inactive metarhodopsin I state.

Animals↗
PubMed1983

Bacteriorhodopsin remains dispersed in fluid phospholipid bilayers over a wide range of bilayer thicknesses.

We have used vesicles made from delipidated bacteriorhodopsin and synthetic lecithins to address the following questions. If the transmembrane dimension of a protein hydrophobic surface differs from the equilibrium thickness of its lipid bilayer environment, will protein monomers aggregate to decrease the protein-lipid contact surface area? If so, how large must the difference be to induce aggregation? Using lecithins with acyl chains from di-10:0 to di-24:1, the thickness of the bilayer hydrocarbon region above the lipid phase transition temperature (tm) was varied from 14.5 A less than to 7.5 A more than the transmembrane dimension of the bacteriorhodopsin hydrophobic region. Bacteriorhodopsin remains dispersed when the surrounding bilayer hydrophobic region is 4 A thicker or 10 A thinner than the bacteriorhodopsin hydrophobic surface. Only the thin- (10:0) and thick- (24:1) bilayer samples showed any bacteriorhodopsin aggregation above tm. Thus a surprisingly large difference between protein and lipid hydrophobic thicknesses can be accommodated without protein aggregation. The lipid bilayer can evidently sustain large local distortions with a small change in free energy.

Bacteriorhodopsins↗
PubMed1999

The structure of bacteriorhodopsin: an emerging consensus.

Six different sets of coordinates have been recently published for bacteriorhodopsin, with reported resolutions ranging from 3.5 A to 2. 3 A. Three of these are the result of electron crystallographic investigations of two-dimensional crystals of bacteriorhodopsin, whereas the others are from X-ray crystallographic studies of three-dimensional crystals of bacteriorhodopsin. How similar are these models? Are the structure determinations using X-ray diffraction data more accurate than those determined by electron crystallography? Is any one of these coordinate sets closer to the 'real' structure of bacteriorhodopsin than the others? Does the availability of newer models bring us closer to understanding how bacteriorhodopsin really works? These questions, as well as some related issues, are currently being explored.

Bacteriorhodopsins↗
PubMed2002

All-trans to 13-cis retinal isomerization in light-adapted bacteriorhodopsin at acidic pH.

The flash photolysis kinetic spectra of the intermediate M(412) of bacteriorhodopsin were monitored during the process of acid titration. In the light-adapted state, the maximum peak amplitude of M(412) absorbance of bacteriorhodopsin decreased (pK(a)=3.40+/-0.05) as the pH decreased from 7.3 to 1.9. In the dark-adapted state, the maximum peak amplitude of M(412) absorbance of bacteriorhodopsin increased as the pH decreased from 6.9 to 4.1, and then decreased (pK(a)=2.85+/-0.05) as the pH dropped to 2.1. These different trends in the change in the maximum peak amplitude suggested that not only the transition of purple membrane to blue membrane had taken place in both light and dark-adapted states, but also the fraction of all-trans-bR had changed during the acid titration. The pH-dependent absorption changes at 640 nm of bacteriorhodopsin in both light- and dark-adapted states were also observed. The pK(a)-values of the purple-to-blue transition were 3.80+/-0.05 in light-adapted state and 3.40+/-0.05 in dark-adapted state, respectively. According to Balashov's method, the fraction of all-trans-bR was assayed as the pH decreased. All these results indicated that the purple-to-blue transition of light-adapted bacteriorhodopsin was accompanied by an all-trans to 13-cis retinal isomerization at acidic pH.

Bacteriorhodopsins↗
PubMed1991

Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction.

Bacterioopsin, expressed in Escherichia coli as a fusion protein with 13 heterologous residues at the amino terminus, has been purified in the presence of detergents and retinylated to give bacteriorhodopsin. Further purification yielded pure bacteriorhodopsin, which had an absorbance ratio (A280/A lambda max) of 1.5 in the dark-adapted state in a single-detergent environment. This protein has a folding rate, absorbance spectrum, and light-induced proton pumping activity identical with those of bacteriorhodopsin purified from Halobacterium halobium. Protein expressed from the mutants D85N, D96N, and R82Q and purified similarly yielded pure protein with absorbance ratios of 1.5. Proton pumping rates of bacteriorhodopsins with the wild-type sequence and variants D85N, D96N, and R82Q were determined in phospholipid vesicles as a function of pH. D85N was inactive at all pH values, whereas D96N was inactive from pH 7.0 to pH 8.0, where wild type is most active, but had some activity at low pH. R82Q showed diminished proton pumping with the same pH dependence as for wild type. Bacteriorhodopsin purified from E. coli crystallized in two types of two-dimensional crystal lattices suitable for low-dose electron diffraction, which permit detailed analysis of structural differences in site-directed variants. One lattice was trigonal, as in purple membrane, and showed a high-resolution electron diffraction pattern from glucose-sustained patches. The other lattice was previously uncharacterized with unit cell dimensions a = 127 A, b = 67 A, and symmetry of the orthorhombic plane group pgg.

Bacteriorhodopsins↗
PubMed1991

High-resolution 13C NMR study of the topography and dynamics of methionine residues in detergent-solubilized bacteriorhodopsin.

The proton transport membrane protein bacteriorhodopsin has been biosynthetically labeled with [methyl-13C]methionine and studied by high-resolution 13C NMR after solubilization in the detergent Triton X-100. The nine methionine residues of bacteriorhodopsin give rise to four well-resolved 13C resonances, two of which are shifted upfield or downfield due to nearby aromatic residues. Methionine residues located on the hydrophilic surfaces, on the hydrophobic surface, and in the interior of the protein could be discriminated by studying the effects of papain proteolysis, glycerol-induced viscosity increase, and paramagnetic broadening by spin-labels on NMR spectra. Such data were used to evaluate current models of the bacteriorhodopsin transmembrane folding and tertiary structure. T2 and NOE measurements were performed to study the local dynamics of methionine residues in bacteriorhodopsin. For the detergent-solubilized protein, hydrophilic and hydrophobic external residues undergo a relatively large extent of side chain wobbling motion while most internal residues are less mobile. In the native purple membrane and in reconstituted bacteriorhodopsin liposomes, almost all methionine residues have their wobbling motion severely restricted, indicating a large effect of the membrane environment on the protein internal dynamics.

Amino Acid Sequence↗
PubMed1981

Linear relations between proton current and pH gradient in bacteriorhodopsin liposomes.

The dependence of proton movement across the membrane of bacteriorhodopsin liposomes on the pH gradient was investigated. Under the appropriate experimental conditions, endogenous proton (or hydroxyl) leakage, proton movement catalyzed by protonophore or nigericin, and light-driven proton translocation depend linearly on the pH gradient. This justifies the use of linear proton flux vs. protonmotive force relations in a recent mosaic thermodynamic description of ion translocation in bacteriorhodopsin liposomes [Westerhoff, H. V., Scholte, B. J., & Hellingwerf, K. J. (1979) Biochim. Biophys. Acta 547, 544-560]. Since bacteriorhodopsin liposomes are a model system for all biological energy transducing systems in which proton pumps are involved, these findings also explain linear relations between proton flux and protonmotive force observed in and postulated for those systems. In cases where the membrane potential is not clamped at a low value, an initial phase of rapid proton movement occurs, followed by a phase of slower proton movement. The rate of proton movement during the slow phase is again linear with the pH gradient. Such a linear relation is not observed for the fast phase. Since the rapid proton movement phase is also observed in liposomes without bacteriorhodopsin, it is not due (only) to dissociation of scalar protons from bacteriorhodopsin. We suggest that during the initial phase of proton movement, the proton flux is not yet electrically compensated by the fluxes of other ions.

Bacteriorhodopsins↗
PubMed1981

Calorimetric and fluorescence depolarization studies on the lipid phase transition of bacteriorhodopsin--dimyristoylphosphatidylcholine vesicles.

The thermotropic lipid phase transition of dimyristoylphosphatidylcholine vesicles reconstituted with bacteriorhodopsin was investigated as a function of the lipid to protein ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. Two attractive features of this system are that the lipid phase transition induces lipid-protein segregation and that the state of aggregation of the protein is known. Above the lipid phase transition and above molar lipid to protein ratios of about 100, bacteriorhodopsin is monomeric. Well below the phase transition, bacteriorhodopsin is aggregated in a hexagonal protein lattice. With increasing amounts of incorporated bacteriorhodopsin, the calorimetric transition broadens, and a second component develops at a temperature which is lower than that of the unperturbed transition. The latter transition was assigned to the disaggregation of the bacteriorhodpsin lattice which occurs 6-7 degrees C below the phase transition of the protein-free lipids according to previous measurements. The van't Hoff enthalpy of the phase transition, as determined from the fluorescence depolarization of diphenylhexatriene, is in surprisingly good agreement with that obtained from differential scanning calorimetry over a wide range of lipid to protein ratios. The differential scanning calorimetry data can be simulated on the basis of a model which takes the protein segregation and crystallization specifically into account. The essential feature of this model is that calorimetrically detectable lipid melting occurs in the temperature region of the protein crystallization, since for the disaggregation of the close packed bacteriorhodopsin lattice additional lipids of an intermediate chain conformation are required.

Bacteriorhodopsins↗
PubMed1999

Structural change of threonine 89 upon photoisomerization in bacteriorhodopsin as revealed by polarized FTIR spectroscopy.

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.

Aspartic Acid↗
PubMed1980

Light isomerizes the chromophore of bacteriorhodopsin.

The primary photochemical event in the two light-transducing pigments whose chromophore is retinal, rhodopsin or bacteriorhodopsin, is a source of controversy. It was originally proposed that the primary photoevent in the bleaching of rhodopsin is the photoisomerization of the chromophore from 11-cis to all-trans retinal. Photochemical considerations suggested that a photoisomerization is the primary event in both rhodopsin and bacteriorhodopsin. However, this description of bacteriorhodopsin's photochemistry has been questioned. To elucidate this problem, we determined the isomeric conformation of retinal for two of the photolytic intermediates of bacteriorhodopsin, using a method that enables us to extract chromophores from the photocycle intermediates L and M at low temperatures (-74 degrees C), and have determined the isomeric conformation of the extracted retinals by HPLC. Here we provide direct evidence that isomerization of the chromophore has taken place in two of the early photocycle intermediates (L and M) of bacteriorhodopsin.

Bacteriorhodopsins↗