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PubMed1979

Time-resolved resonance Raman characterization of the bO640 intermediate of bacteriorhodopsin. Reprotonation of the Schiff base.

The resonance Raman spectrum of photolyzed bacteriorhodopsin under conditions known to increase the concentration of the bO640 intermediate in both H2O and D2O is presented. By use of computer subtraction techniques and a knowledge of the Raman spectra of the unphotolyzed bacteriorhodopsin as well as the other intermediates in the cycle, a qualitative spectrum of bO640 is determined. The shift of a band at 1630 cm-1 in H2O to 1616 cm-1 in D2O suggests that the Schiff base of bO640 is protonated. Additional bands at 947, 965, and 992 cm-1 that appear only in D2O suspensions confirm that a proton is coupled to the retinal chromophore of bO640. The reprotonation of the Schiff base thus occurs during the bM412 to bO640 step. The fingerprint region, sensitive to the isomeric configuration of the retinal chromophore of bO640, is dissimilar to the fingerprint regions of published model compounds and other forms of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1977

Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

A simple technique is described that uses a continuous wave laser with electromechanical modulation to obtain time-resolved Raman spectra of transient species on the millisecond timescale. The time behavior of the vibrational bands of the intermediates involved in the proton pumping of bacteriorhodopsin is determined. From these results, along with resonance enhancement and power dependence studies, the bands that appear in the continuous wave Raman spectrum of bacteriorhodopsin can be assigned to three intermediates in the photochemical cycle of bacteriorhodopsin, bR570, bL550, and bM412. The Raman spectra of bR570 and bM412 are compared with published spectra of model Schiff bases of all-trans and 13-cis retinal.

Bacteriorhodopsins↗
PubMed1977

Orientation of bacteriorhodopsin in Halobacterium halobium as studied by selective proteolysis.

The orientation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by proteolytic degradation of purple membrane sheets, reconstituted vesicles, and whole cells, with the following results: (i) Bacteriorhodopsin in purple membrane sheets is cleaved at a single site by Pronase or trypsin; a polypeptide segment of about 15 amino acids is lost from the carboxyl end. Carboxypeptidase A sequentially releases amino acids from the carboxyl end; the tetrapeptide sequence -Ala-Ala-Thr-Ser(COOH) was tentatively deduced for this terminus. (ii) The apomembrane, which lacks retinal, undergoes a second cleavage with trypsin releasing a fragment of approximately 6300 molecular weight from the amino terminus. (iii) Vesicles reconstituted from the purple membrane sheets and synthetic lecithins, in which the direction of proton pumping is opposite to that in the whole cells, have the carboxyl terminus of bacteriorhodopsin accessible to proteolysis. (iv) In envelope vesicles, which largely pump protons in the same direction as the whole cells, the carboxyl terminus is largely protected against proteolysis. (v) Treatment of whole cells with proteinase K hydrolyzes the cell wall proteins but has no effect on acteriorhodopsin. However, the same treatment after lysis of the cells results in degradation of the hydrophilic region at the carboxyl terminus. The results show that the carboxyl terminus as well as the additional cleavage site near the amino terminus observed in apomembrane are on the cytoplasmic side of the purple membrane.

Apoproteins↗
PubMed1979

Photoisomerization, energy storage, and charge separation: a model for light energy transduction in visual pigments and bacteriorhodopsin.

A simple model for the early events in visual pigments and bacteriorhodopsin is proposed. The model makes use of the likelihood that a negatively charged amino acid forms a salt bridge with the positively charged nitrogen of the retinylic chromophore. The photochemical event is a cis-trans isomerization in visual pigments and a trans-cis isomerization in bacteriorhodopsin, which in each case cleaves the salt bridge and thus separates charge in the interior of the protein. We propose that this is how the energy of a photon is transduced into chemical free energy of the primary photoproduct. The use of photoisomerization of a flexible chromophore to achieve charge separation provides a general mechanism which may be applicable to other systems. Our model explains many of the fundamental properties of visual pigments and their photoproducts. First, the extraordinarily low rate of thermally populating the ground state of the primary photoproduct, as determined from psychophysical and electrophysiological measurements, is seen as resulting from the large barrier to thermal isomerization about a double bond, perhaps enhanced by electrostatic attraction in the salt bridge. Second, the increase in energy and the spectral red shift that characterize the primary photochemical events are natural consequences of the separation of charge. Proton-dependent processes detected with picosecond techniques are proposed to be ground-state relaxation processes following the primary photochemical event. Finally, the charged groups of the salt bridge, repositioned by photoisomerization, provide a simple mechanism for vectorial proton translocation in bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1978

Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments.

Pronase treatment of aqueous suspensions of purple membrane fragments from H.halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000--21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme. Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated. Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M412 and the regeneration of bR570. Simultaneously the yield of O660 is reduced. As with untreated purple membrane, the correlation between the rates of decay of M412 and regeneration of bR570 is greatest when the yield of O660 is lowest.

Bacterial Proteins↗
PubMed1979

Bacteriorhodopsin in liposomes. I. A description using irreversible thermodynamics.

A comprehensive description of light-induced ion transport in bacteriorhodopsin liposomes is presented. Linear irreversible thermodynamics and the chemiosmotic theory serve as theoretical bases for the formulation of a limited number of fundamental equations. In these equations mechanistic parameters characterize the dependence of ion movement and flux through the photochemical cycle of bacteriorhodopsin on electrochemical potential differences and a so-called light affinity. By making appropriate steady-state assummptions and carrying out mathematical reduction experimentally testable expressions, still containing the mechanistic parameters, are obtained. In the accompanying article rigid trials to falsify these expressions are shown to be unsuccessful.

Bacteriorhodopsins↗
PubMed1979

Bacteriorhodopsin (BR570) bathochromic band shift in an external electric field.

In dry films of bacteriorhodopsin-containing purple membranes from Halobacterium halobium the external electric field (10(4) -- 10(5) V . cm-1) induces the appearance of a product spectrally close to the initial intermediate of bacteriorhodopsin (BR) photochromic cycle (bathoform, K). This result and also preliminary data of the electret-thermal analysis of the preparations suggest that the dielectric polarization in chromophore-protein-lipid complexes might be an essential step of the primary stabilization of light energy in photo-bioenergetic processes.

Bacteriorhodopsins↗
PubMed1978

Investigation by focused laser beam scanning of the photoelectric activity of bacteriorhodopsin-containing lipid bilayers.

The photoelectric activity of different parts of lipid bilayer containing bacteriorhodopsin was investigated by moving a small actinic light spot across the Plateau-Gibbs border and the bimolecular part of this reconstituted model membrane. The results give direct evidence that bacteriorhodopsin incorporated into the bimolecular region of the lipid membrane is responsible for the photoelectric activity of this system. A technique for scanning the photoelectric activity of a modified bimolecular lipid membrane is described in detail.

Bacteriorhodopsins↗
PubMed1978

Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.

Bacteriorhodopsins↗
PubMed1979

Time-resolved resonance Raman characterization of the bL550 intermediate and the two dark-adapted bRDA/560 forms of bacteriorhodopsin.

The resonance Raman spectrum of the second intermediate in the bacteriorhodopsin cycle, bL550, is obtained by a simple flow technique. The Schiff base linkage in this intermediate appears to be protonated, contrary to previous suggestion. The fingerprint region of the spectrum of bL550 does not closely match those of any presently available model Schiff bases of retinal isomers, though some comparisons can be made. The resonance Raman spectrum of dark-adapted bacteriorhodopsin is obtained and decomposed by computer subtraction of the spectrum of bR570. The remaining spectrum does not match the spectra of any model compounds presently in the literature. The spectra of bL550 and dark-adapted bRDA/560 from purple membrane in H2O are compared to those in D2O. It is found that changes in the spectrum occur in the 1,600 - 1,650 cm-1 region as well as in the 800 - 1,000 cm-1 region, but apparently not in the fingerprint region (1,100 - 1,400 cm-1). The possibilities of conformational changes of the retinal chromophore in the light adaptation process as well as the photosynthetic cycle are discussed.

Bacteriorhodopsins↗
PubMed1979

The quantum yield of flash-induced proton release by bacteriorhodopsin-containing membrane fragments.

The quantum yield of proton release by bacteriorhodopsin was measured from volume changes after excitation of purple membrane fragments by short flashes. At low ionic strengths, about 0.25 mol of protons is released per einstein absorbed. This agrees well with quantum yields reported recently for the conversion of bacteriorhodopsin into a metastable state (M) that absorbs near 412 nm. However, the quantum yield of proton release increases gradually with increasing ionic strength; it plateaus with a value of 0.43 +/- 0.03 at ionic strengths above 200 mM. Changing the ionic strength has no detectable effect on the quantum yield of formation of the M spectral state. It thus appears that as many as two protons can be released and rebound in each photochemical cycle at high ionic strengths. The quantum yield of proton release is essentially independent of pH over the range 6.0-8.75. The quantum yield decreases with increasing flash strength, apparently due to photoreversal of the initial photochemical reaction.

Bacteriorhodopsins↗
PubMed1979

Bacteriorhodopsin in model membranes. A new component of the displacement photocurrent in the microsecond time scale.

A quasi-short-circuit (tunable voltage clamp) measurement method with microsecond time resolution was applied to a bacteriorhodopsin model membrane formed by a novel interfacial technique. A new component (B1) of the displacement photocurrent was recorded: it has no detectable latency at an instrumental time constant of 1.5 museconds, and persists at 5 degrees C. In addition, a slower component (B2) of opposite polarity inhibited by low temperature (5 degrees C) and low pH (pH = 3.0) was recorded. The technique is very sensitive for the study of fast capacitative photoresponses in model membranes, and allows the detection of charge displacements in bacteriorhodopsin associated with distinct stages of the photochemical transformation.

Bacteriorhodopsins↗
PubMed1977

Molecular weight of bacteriorhodopsin solubilized in Triton X-100.

Bacteriorhodopsin from Halobacterium halobium has been solubilized in the nonionic detergent Triton X-100. The circular dichroic spectrum and hydrodynamic properties indicate that the structure of this protein in the detergent is not significantly altered from that of the native membrane-bound form. Bacteriorhodopsin is monomeric under the conditions of solubilization with a molecular weight of 24,250+/-2,000 and binds approximately one micelle of Triton X-100.

Bacteriorhodopsins↗
PubMed1979

Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins.

The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin have been determined. These results are in agreement with those recently published by Ovchinnikov and coworkers [Ovchinnikov, Y.A., Abdulaey, N.G., Feigina, M.Y., Kiselev, A.V. & Lobanov, N.A. (1977) FEBS Lett. 84, 1-4]. Chymotryptic cleavage of bacteriorhodopsin produced two fragments, C-1 (Mr 19,000) and C-2 (Mr 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.

Amino Acid Sequence↗
PubMed1978

A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin.

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.

Amino Acids↗
PubMed1977

The photochemical cycle of bacteriorhodopsin.

The reaction cycle of bacteriorhodopsin in the purple membrane isolated from Halobacterium halobium has been studied by optical absorption spectroscopy using low-temperature and flash kinetic techniques. After absorption of light, bacteriohodopsin passes through at least five distinct intermediates. The temperature and pH dependence of the absorbance changes suggests that branch points and/or reversible steps exist in this cycle. Flash spectroscopy in the presence of a pH-indicating dye shows that the transient release of a proton accompanies the photoreaction cycle. The proton release occurs from the exterior and the uptake is on the cytoplasmic side of the membrane, as required by the function of bacteriorhodopsin as a light-driven proton pump. Proton translocating steps connecting release and uptake are indicated by deuterium isotope effects on the kinetics of the cycle. The rapid decay of a light-induced linear dichroism shows that a chromophore orientation change occurs during the reaction cycle.

Bacteriorhodopsins↗
PubMed1977

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin.

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF1) as well as several hydrophobic compoenents. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yield vesicles that catalyzed light-dependent ATP formation. Both the 32Pi-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2'-dichloro-4'-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-beta-D-glucopyranosyl-4,6'-dihydroxydihydrochalcone were also effective inhibitors of both reactions.

Adenosine Triphosphatases↗