O-H stretching vibration in Fourier transform difference infrared spectra of bacteriorhodopsin.

FTIR difference spectroscopic studies of M intermediate and LA bacteriorhodopsin in the O-H stretching region show bands at 3671 and 3641 cm-1, respectively. The O-H stretching bands in this region may reflect protonation-deprotonation changes or environmental change in the tyrosine residues in bR.

Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states.

Solid-state 13C NMR spectra were employed to characterize the protonation state of tyrosine in the light-adapted (bR568) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for [4'-13C]Tyr-labeled bR, regenerated with [14-13C]retinal as an internal marker to identify the photocycle states. The [14-13C]retinal has distinct chemical shifts for bR555, for bR568, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate are easily distinguished on the basis of the chemical shift of the 4'-13C label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, we show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at -80 degrees C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. In addition, after the M samples were briefly warmed (approximately 30 s), no tyrosinate was observed. However, small (1-2 ppm) changes in the structure or dispersion in the Tyr peak were observed in the M state phototrapped by either method. These changes were reversible when the sample was warmed, although on a time scale slower than the relaxation of the retinal back to the bR568 conformer.(ABSTRACT TRUNCATED AT 250 WORDS)

The photocycle of bacteriorhodopsin immobilized in poly(vinyl alcohol) film.

The kinetics of photoelectric and optical signals were measured on samples containing oriented purple membranes immobilized in a poly(vinyl alcohol) film and on purple membranes introduced into a PVA-H2O mixture. The bacteriorhodopsin photocycle in the PVA-H2O mixture was complete. The only observed changes were the slowing down of the optical and electrical signals in relation to the M412-O640 and O640-bRall-trans steps. In the PVA film the O640 intermediate disappeared and a negative photoelectric signal appeared.

Conformational changes in sensory rhodopsin I: similarities and differences with bacteriorhodopsin, halorhodopsin, and rhodopsin.

FTIR difference spectra have been obtained for the sR587----S373 phototransition of sensory rhodopsin I (sR-I), a signal-transducing protein of Halobacterium halobium. The vibrational modes of the sR587 chromophore have frequencies close to those of the bacteriorhodopsin bR568 chromophore, confirming that the two chromophores have very similar structures and environments. However, the sR-I Schiff base C = N stretch frequency is downshifted relative to bR, consistent with weaker hydrogen bonding with its counterion(s). The carboxyl (COOH) stretch modes of sR-I and halorhodopsin (hR) are at the same frequencies. On the basis of sequence homologies, these bands can be assigned to Asp-106 in helix D and/or Asp-201 in helix G. In contrast, no band was found that could be assigned to the protonation of Asp-76. In bR, the homologous residue Asp-85 serves as the acceptor group for the Schiff base proton. Bands appear in the amide I and II regions at similar frequencies in sR-I, hR, and bR, indicating that despite their different functions they all undergo closely related structural changes. Bands are also detected in the C-H stretch region, possibly due to alterations in the membrane lipids. Similar spectral features are also observed in the lipids of rhodopsin-containing photoreceptor membrane upon light activation.

The interaction between halogenated anaesthetics and bacteriorhodopsin in purple membranes as examined by intrinsic ultraviolet fluorescence.

In the presence of halogenated general anaesthetics such as enflurane and halothane, the spectral properties of the bacteriorhodopsin pigment contained in the purple membranes of Halobacterium halobium are strongly modified. It is reversibly transformed into a red-coloured species absorbing maximally at 480 nm, at the expense of its characteristic 570-nm absorption band. The ultraviolet fluorescence of bacteriorhodopsin has been used to probe the structural modifications that are reflected by this spectral change. Our results show that they are very small and do not perturb the energy transfer dynamics which take place between the aromatic amino acid residues and the retinyl chromophore. The fluorescence properties of anaesthetic-treated bacteriorhodopsin are dominated by the quenching properties of the halogenated hydrocarbon, which are obvious even at anaesthetic concentrations under those needed to induce a spectral change in the bacteriorhodopsin chromophore. This does not rule out direct interaction between anaesthetics and bacteriorhodopsin, but it indicates that the chromophoric site might well not be their primary target.

Effect of external salinity changes on cellular composition of some ions and amino acids in Deleya halophila.

Changes in total cellular composition of some ions and amino acids of the moderate halophile Deleya halophila were studied in response to external salinity changes. Among the 14 amino acids investigated, the cellular glycine and aspartic acid content increased with increasing salinity. D. halophila also accumulated Na+ at the highest external salt concentrations.

Fast atom bombardment mass spectrometry as a rapid means of screening mixtures of ether-linked polar lipids from extremely halophilic archaebacteria for the presence of novel chemical structures.

Fast atom bombardment mass spectrometry was used to analyse intact polar ether lipids present at microgram levels in crude lipid mixtures extracted from Halobacterium halobium, Natronococcus occultus and Halobacterium marismortui. Negative-ion spectra showed the intact deprotonated lipid molecules and in some instances their sodium salts. The simplicity of the mass spectra permits the rapid screening of polar lipid mixtures for the presence of novel lipids. Additional structural information of ions with selected masses was obtained after collisionally induced decomposition.

X-ray diffraction and flash photolysis studies of M intermediate of lattice-contracted purple membrane.

The effects of cross-linking and lattice contraction of purple membrane (PM) on the photodynamics of bacteriorhodopsin (bR) and on the tertiary structure were studied by flash photolysis and X-ray diffraction. To get a contracted lattice form of PM, native PM, and/or PM cross-linked by glutaraldehyde were treated with deoxycholate or Triton X-100. Part of the Triton-treated cross-linked PM was further incubated with Bio-Beads SM-2 to remove Triton X-100. In the modified PM, several long-lived components of the M intermediate appeared, the features of which were related to the environment of bR. Also, X-ray diffraction studies using synchrotron radiation were performed on the modified PM under intense light irradiation (lambda greater than 500 nm) in which 40-80% of bR was photoconverted to the M state. In the Triton-treated cross-linked PM dispersed in 0.25% Triton X-100, the unit cell of membrane crystalline lattice was enlarged from 58.8 to 59.8 A and the crystalline order decreased with irradiation. The analysis of X-ray diffraction patterns suggests that light-induced conformational changes of bR correlated with the Triton content of the environment and an increase of substitution disorder was caused by these changes, but the average location of bR was unchanged. However, the other modified PM showed no significant changes of diffraction, upon light irradiation.

Regeneration of bacteriorhodopsin in mixed micelles.

Regeneration of bacteriorhodopsin from bacterioopsin and all-trans-retinal was studied in a mixed micelle system consisting of dodecyl sulfate, CHAPS and a water-soluble phospholipid dihexanoylphosphatidylcholine (hex2-PhosChol). Regeneration to approximately 40,000 M-1.cm-1 extinction at 550 nm (epsilon 550) was obtained with either 2.3 mM or 6.5 mM CHAPS along with 6.9 mM dodecyl sulfate and 4.5 mM hex2-PhosChol in 0.16 M NaCl and 40 mM phosphate (pH 6.0). Without CHAPS, the regeneration in 4.5 mM Hex2-PhosChol gave epsilon 555 = 27,800; without PhosChol, the 1:3 CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 20,000; and without PhosChol the nearly equimolar CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 10,000. The composition of the mixed micelles was estimated from fluorescence spectroscopy using pyrene butyryl hydrazine. The molecular weight was estimated by molecular seive chromatography to be 87,100 for 2.3 mM CHAPS, 6.9 mM dodecyl sulfate and 0.67 mM hex2-PhosChol; and 83,200 for 7.0 mM CHAPS, 6.9 mM dodecyl sulfate, and 1.1 mM hex2-PhosChol. These results are consistent with the idea that at low concentrations of CHAPS and dodecyl sulfate, CHAPS organizes the dodecyl sulfate into disk shaped bilayer micelles that are favorable for bacterioopsin refolding. However, a high concentration of either detergent inhibits regeneration. Added hex2-PhosChol can overcome the inhibitory effects of high concentrations of either CHAPS or dodecyl sulfate.

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent.

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [125I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes from Halobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[14C]-cyanate binding to BR. [125I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generated in situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.

The role of back-reactions and proton uptake during the N----O transition in bacteriorhodopsin's photocycle: a kinetic resonance Raman study.

The kinetics of bacteriorhodopsin's photocycle have been analyzed at pH 5, 6, 7, 8, and 8.6 by using time-resolved resonance Raman spectroscopy. The concentrations of the various intermediates as a function of time were determined by following their resonance Raman intensities using 502-nm (L550, N550, BR568), 458-nm (M412), and 752-nm (O640) excitation. The spectral contributions to the pump + probe data from each intermediate were quantitatively separated by least-squares decomposition. These relative concentrations were then converted to absolute concentrations by using a conservation of molecules constraint. This enabled the unambiguous refinement of a variety of kinetic models to find the simplest one that accurately describes the data. The kinetic data, including the biphasic decay of L550 and M412, are best reproduced by a sequential scheme including back-reactions (BR----L----M----N----O----BR). In addition, the kinetics of the L----M and N----O steps are found to be pH-dependent. Both the forward and reverse rate constants connecting L550 and M412 increase with pH, confirming earlier proposals of catalyzed Schiff base deprotonation at alkaline pH. Below pH 7, the N550----O640 rate constant is independent of pH, but it decreases linearly with pH above 7. This indicates that the protein must pick up a proton during the N550----O640 transition and that this process becomes rate determining above pH 7. There must, therefore, be an intermediate between N550 and O640 which we denote as N+550. A molecular graphics model is presented which incorporates these observations into a mechanism for proton pumping.

Light- and dark-adapted bacteriorhodopsin, a time-resolved neutron diffraction study.

Recently, neutron diffraction experiments have revealed well-resolved and reversible changes in the protein conformation of bacteriorhodopsin (BR) between the light-adapted ground state and the M-intermediate of the proton pumping photocycle (Dencher, Dresselhaus, Zaccai and Büldt (1989) Proc. Natl. Acad. Sci. USA 86, 7876-7879). These changes are triggered by the light-induced isomerization of the chromophore retinal from the all-trans to the 13-cis configuration. Dark-adapted purple membranes contain a mixture of two pigment species with either the all-trans- or 13-cis-retinal isomer as chromophore. Employing a time-resolved neutron diffraction technique, no changes in protein conformation in the resolution regime of up to 7 A are observed during the transition between the two ground-state species 13-cis-BR and all-trans-BR. This is in line with the fact that the conversion of all-trans BR to 13-cis-BR involves an additional isomerization about the C15 = N Schiff's base bond, which in contrast to M formation minimizes retinal displacement and keeps the Schiff's base in the original protein environment. Furthermore, there is no indication for large-scale redistribution of water molecules in the purple membrane during light-dark adaptation.

Nucleotide sequence of four genes encoding ribosomal proteins from the 'S10 and spectinomycin' operon equivalent region in the archaebacterium Halobacterium marismortui.

Four genes encoding ribosomal proteins HmaS17, HmaL14, HmaL24 and HS3, have been identified in the lambda EMBL3 clone PP*7 from a genomic library of the archaebacterium Halobacterium marismortui. The clone contains genes from the 'S10 and spectinomycin' operon equivalent region. Three of the deduced proteins are homologous to the corresponding Escherichia coli and Methancoccus vannielii S17, L14 and L24 proteins, as well as to eukaryotic proteins from rat or yeast. HS3 was identified as an extra protein corresponding to the gene product for orfc in M. vannielii and the eukaryotic ribosomal protein RS4 from rat. The equivalence of HmaL24 (HL16) and E. coli L24, which share only 28% identical amino acid residues, could now be shown by localizing the HmaL24 gene at the same position in the cluster.

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing.

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.

Analysis of high-resolution electron diffraction patterns from purple membrane labelled with heavy-atoms.

Progress in the structure determination of bacteriorhodopsin, the protein component of purple membrane from Halobacterium halobium has been limited by the lack of three-dimensional phase information between 6 and 3 A resolution. By analogy with X-ray methods, it is possible that heavy-atom labelling of the membrane crystal may provide heavy-atom derivatives that can be used for phasing by the multiple isomorphous replacement method. This paper describes the screening of heavy-atom compounds as potential derivatives, and the evaluation of the data collected from these heavy-atom-labelled membranes. Improvements in the methods for collecting electron diffraction data and analysing and merging the data are presented. Diffraction patterns of purple membrane samples were taken at -120 degrees C to minimize radiation damage. About 30 heavy-atom compounds were tested for use as potential derivatives. The diffraction patterns from labelled membranes were analysed by examining 6.5 A difference Fourier maps. Two heavy-atom compounds were selected for three-dimensional data collection at 3 A resolution. In addition, a full set of native data at -120 degrees C was collected to 2.7 A resolution. The intensity merging, heavy-atom derivative evaluation, heavy-atom refinement and the calculation of phases are presented. Phases are compared to those determined by electron microscope imaging, and limitations of the method are discussed. It is concluded that, with the present accuracy of data collection and the present magnitude of delta F/F available for the derivatives, the phasing power is too small. The phases that are obtained are not sufficiently accurate to provide a reliably interpretable map. It may be possible, however, to use the heavy-atom derivative data in difference Fourier calculations in which the presence of a peak would confirm the phases calculated from a model or obtained by electron microscope imaging.

Two-dimensional maps in very acidic immobilized pH gradients.

Up to the present time it has been impossible to perform two-dimensional (2-D) separations in very acidic immobilized pH gradients (IPG), due to the lack of suitable buffering acrylamido derivatives to be incorporated into the polyacrylamide matrix. The advent of the pK 3.1 buffer (2-acrylamido glycolic acid; Righetti et al., J. Biochem. Biophys. Methods 16, 1988, 185-192) allowed the formulation of such acidic gradients. We report here separations in IPG pH 2.8-5.0 intervals of polypeptide chains from total lysates of rat intestinal and liver cells and 30S and 50S ribosomal proteins from Halobacterium marismortui. Conditions are given for highly reproducible first and second dimension gels and for a proper silver staining of 2-D maps with practically no background deposition.

Measuring changes in membrane thickness by scanning tunneling microscopy.

We investigated the feasibility of using the scanning tunneling microscope (STM) as a morphometric tool to measure the thickness of biomembranes. Planar monolayers of oriented purple membrane (PM) were prepared, nitrogen-dried or freeze-etched, and coated with metal. PM thickness was quantified by STM and transmission electron microscopy. STM calibration and the effect of contamination-mediated surface deformation on measurements of PM thickness were evaluated. The thickness of PM attached to mica and glass and the effect of papain on PM thickness were also examined. The apparent thickness of enzymatically modified PM increased after papain treatment. The mean thickness of both nitrogen-dried PM on mica and freeze-etched PM on glass was 4.6 nm. After papain treatment PM thickness on mica increased to 4.8 nm and on glass to 5.4 nm. These results demonstrate that STM analysis of metal-coated planar membrane monolayers can be used to measure changes in average membrane thickness at sub-nanometer resolution.

Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism.

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).

The interaction of Triton X-100 with purple membranes. Detergent binding, spectral changes and membrane solubilization.

The interaction of the non-ionic surfactant Triton X-100 with Halobacterium purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a half-time of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent-mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built-in probe to monitor detergent-induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from all-trans to 13-cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X-100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal-bacteriorhopsin Schiff's base (bleaching).

Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin.

The effect of lipid-protein interaction on the photodynamics of bacteriorhodopsin (bR) was investigated by using partially delipidated purple membrane (pm). When pm was incubated with a mild detergent, Tween 20, the two major lipid components of pm, phospholipids and glycolipids, were released in different ways: the amount of phospholipids released was proportional to the logarithm of the incubation time; the release of glycolipids became noticeable after the release of approximately 2 phospholipids/bR, but soon leveled off at approximately 50% of the initial content. It was found that the thermal decay of the photocycle intermediate N560 was inhibited by the removal of less than 2 phospholipids per bR. This inhibition was partly explained by an increase in the local pH near the membrane surface. More significant changes in the bR photoreactions were observed when greater than 2 phospholipids/bR were removed: (1) the extent of light adaptation became much smaller, and this reduction correlated with the release of glycolipids; (2) N560 became difficult to detect; (3) the M412 intermediate, which is characterized by a pH-insensitive lifetime, was replaced by a long-lived M-like photoproduct with a pH-sensitive lifetime. The heavy delipidation apparently altered the mechanism by which the deprotonated Schiff base receives a proton. An important conformational change in the protein moiety is suggested to take place during the M412 state, this conformational change being inhibited in the rigid lipid environment.