Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle

The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied. The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium. The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin. The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate. Similar results were also obtained with the Pro-186----Gly substitution. In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate. These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.     

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.     

Quantum efficiency of the photochemical cycle of bacteriorhodopsin

Values in the literature for the quantum efficiency of the photochemical cycle of bacteriorhodopsin (bR) range from 0.25 to 0.79 and the sum of the quantum yields of the forward and back photoreactions [Formula: see text] has been proposed to be 1. In the present work, low intensity laser flashes (532 nm) and kinetic spectroscopy were used to determine the quantum efficiency of bR photoconversion, [UNK]_bR, by measuring transient bleaching of bR at 610 nm in the millisecond time scale. Bovine rhodopsin (R) in 2% ammonyx LO was used as a photon counter. We find that the ratio of the quantum yields of bacteriorhodopsin photoconversion and bleaching of rhodopsin, [UNK]_bR/[UNK]_R, is 0.96 ± 0.04. Based on the quantum yield of the photobleaching of rhodopsin, 0.67, the quantum efficiency of bR photoconversion was determined to be 0.64 ± 0.04. The quantum yield of M formation was found to be 0.65 ± 0.06. From the transient bleaching of bR at 610 nm with a saturating laser flash (28 mJ/cm²) the maximum amount of bR cycling was estimated to be 47 ± 3%. From this value and the spectrum of K published in the literature, the ratio of the efficiencies of the forward and back light reactions, [UNK]₁/[UNK]₂, was estimated to be 0.67 ± 0.06 and so [UNK]₂ ≈ 1 (0.94 ± 0.06). The sum of [UNK]₁ + [UNK]₂ ≈ 1.6. It was found that repeated high-intensity laser flashes (>20 mJ/cm²) irreversibly transformed bR into two stable photoproducts. One has its absorption maximum at 605 nm and the other has a well-resolved vibronic spectrum with maxima at 342, 359 (main peak), and 379 nm. The quantum yield of the formation of the photoproducts is ≈ 10^-4.     

Primary intermediates in the photochemical cycle of bacteriorhodopsin.

Picosecond studies of the primary photochemical events in the light-adapted bacteriorhodopsin, bR570, indicate that the first metastable intermediate K610 is formed with a rise time of 11 ps. Difference spectra obtained at 50 ps after excitation show that K610 is the same species as that trapped in low temperature glasses. A precursor species (S) of the K610 intermediate has been observed which is red shifted with respect to K610 and is formed within the 6-ps time width of the excitation pulse. The formation of the precursor has no observable thermal dependence between 298 degrees and 1.8 degrees K. The formation of K610 has a very low thermal barrier and at very low temperatures, the rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation of K610 has a moderate deuterium isotope effect of kH/kD approximately 1.6 at 298 degrees K and 2.4 at 4 degrees K. The mechanism for formation of K610 is found to involve a rate-limiting proton transfer which occurs by tunneling at low temperatures.     

Kinetic isotope effects in the photochemical cycle of bacteriorhodopsin

Kinetics were determined for the four transients K₅₉₀, L₅₄₀, M₄₁₀, O₆₆₀ of the photochemical cycle of bacteriorhodopsin (BR₅₇₀) both in ¹H₂O and in ²H₂O over a wide temperature range. Breaks in the Arrhenius plots, observed at 25–32 for the longest-lived transients coincide with a transition point in the microviscosity of the membrane as measured by depolarization of an added fluorescent probe. The earliest isotope effect occurs in the decay of L₅₄₀, and is present in the subsequent formation and decay of M₄₁₀ and O₆₆₀. Thus in the light-driven proton pump of BR₅₇₀, proton ejection from the Schiff base correlates with decay of L₅₀₄ and reprotonation occurs with the decay of both M₄₁₀ and O₆₆₀ back to BR₅₇₀.     

Kinetic isotope effects in the photochemical cycle of bacteriorhodopsin.

Kinetics were determined for the four transients K590, L540, M410, O660 of the photochemical cycle of bacteriorhodopsin (BR570) both in 1H2O and in 2H2O over a wide temperature range. Breaks in the Arrhenius plots, observed at 25 degrees-32 degrees for the longest-lived transients coincide with a transition point in the microviscosity of the membrane as measured by depolarization of an added fluorescent probe. The earliest isotope effect occurs in the decay of L540, and is present in the subsequent formation and decay of M410 and O660. Thus in the light-driven proton pump of BR570, proton ejection from the Schiff base correlates with decay of L540 and reprotonation occurs with the decay of both M410 and O660 back to BR570.     

Flash photometric experiments on the photochemical cycle of bacteriorhodopsin

The photochemical reaction cycle of bacteriorhodopsin was investigated by means of flash photometric methods. Three different intermediates with absorption maxima at about 630 nm, 411 nm, and 646 nm could be detected. Kinetic data of the occurrence of these intermediates were obtained from isolated purple membrane in different mediums and from intact halobacteria. An activation energy of 14.1±0.4 kcal·mol⁻¹ and of about 19 kcal·mol⁻¹ for the formation of bacteriorhodopsin 411 and of bacteriorhodopsin 565, resp., was calculated. pH-changes in the medium caused by the reaction cycle of bacteriorhodopsin were detected by use of the pH-indicator bromocresol green.     

Dynamical heterogeneity of specific amino acids in bacteriorhodopsin

Components of biological macromolecules, complexes and membranes are animated by motions occurring over a wide range of time and length scales, the synergy of which is at the basis of biological activity. Understanding biological function thus requires a detailed analysis of the underlying dynamical heterogeneity. Neutron scattering, using specific isotope labeling, and molecular dynamics simulations were combined in order to study the dynamics of specific amino acid types in bacteriorhodopsin within the purple membrane (PM) of Halobacterium salinarum. Motions of leucine, isoleucine and tyrosine residues on the pico- to nanosecond time scale were examined separately as a function of temperature from 20 to 300 K. The dynamics of the three residue types displayed different temperature dependence: isoleucine residues have larger displacements compared to the global PM above 120 K; leucine residues have displacements similar to that of PM in the entire temperature range studied; and tyrosine residues have displacements smaller than that of the average membrane in an intermediate temperature range. Experimental features were mostly well reproduced by molecular dynamics simulations performed at five temperatures, which allowed the dynamical characterisation of the amino acids under study as a function of local environment. The resulting dynamical map of bacteriorhodopsin revealed that movements of a specific residue are determined by both its environment and its residue type.     

Flash kinetic study of the last steps in the photoinduced reaction cycle of bacteriorhodopsin

The reaction cycle of light adapted bacteriorhodopsin (BR) in aqueous purple membrane suspensions was studied by laser flash photolysis at different temperatures (2–49°C) and pH values (3–10). The activation energy for several reaction steps was determined at pH 7.6. The kinetics of O-bacteriorhodopsin (one of the last intermediates in the cycle) were analyzed in some detail and it was found that the simple consecutive reaction scheme M-BR → O-BR → BR may explain the kinetics of O-bacteriorhodopsin as measured at 680 nm. Since the pH change in neutral aqueous suspensions of purple membrane follows a similar kinetics as O-bacteriorhodopsin it is suggested that protons are released during the reaction M-BR → O-BR and taken up again during the reaction O-BR → BR.Another long-lived intermediate, which absorbs to a greater extent than bacteriorhodopsin at 570 nm and less than bacteriorhodopsin at 420 nm, was identified with the strongly fluorescing species, pseudo- or P-bacteriorhodopsin. The decay of P-bacteriorhodopsin in bacteriorhodopsin had an activation energy of only approx. 1.2 kcal/mol, which suggests that the last step of the photocycle is a relaxation around a single bond.At pH 9–10, the simple first-order kinetics of all the intermediates were changed into a kinetics consisting of two first-order decays. This change of kinetics was accompanied by a drastic decrease in the rotational diffusion relaxation time.To explain the results obtained in this work and those of others, a model involving proton uptake and release by the Schiff base nitrogen combined with an isomerization reaction is finally proposed.     

High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations

Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96 Asn; PM 374: Asp96 Gly; PM 384: Asp85 Glu) were grown in a synthetic medium containing (4-¹³C)-Asp. The mutant bacteriorhodopsins labeled with (4-¹³C)-Asp (37%–45%), and owing to the metabolism of Halobacteria also with (11-¹³C)-Trp (50%–100%), were isolated as purple membranes and ¹³C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989 a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85 Glu) has an absorption maximum at 610 run. It can be converted into a purple form (_max = 5.40 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed.Abbreviations CHAPSO 3-(3-cholamidopropyl)-dimethylammonio2-hydroxy-1-propane sulfonate - CP crosspolarization - bR bacteriorhodopsin - FTIR Fourier-transform-infrared - FID free induction decay - IR Infrared - MASS Magic angle sample spinning - NMR Nuclear magnetic resonance - TMS tetramethylsilane This research was supported by the Deutsche Forschungsgemeinschaft #SFB 60G9 Offprint requests to: M. Engelhard     

Volume and enthalpy changes in the early steps of bacteriorhodopsin photocycle studied by time-resolved photoacoustics.

We have studied the photoinduced volume changes, energetics, and kinetics in the early steps of the bacteriorhodopsin (BR) photocycle with pulsed, time-resolved photoacoustics. Our data show that there are two volume changes. The fast volume change ( < or = 200 ns) is an expansion (2.5 +/- 0.3 A3/molecule) and is observed exclusively in the purple membrane (PM), vanishing in the 3-[(3-cholamidopropyl)-dimethylammonio] -1-propane-sulfonate-sulfonate-solubilized BR sample; the slow change (approximately 1 micros) is a volume contraction (-3.7 +/- 0.3 A3/molecule). The fast expansion is assigned to the restructuring of the aggregated BR in the PM, and the 1-micros contraction to the change in hydrogen bonding of water at Asp 212 (Kandori et al. 1995. J. Am. Chem. Soc. 117:2118-2119). The formation of the K intermediate releases most of the absorbed energy as heat, with delta Hk = -36 +/- 8 kJ/mol. The activation energy of the K --> L step is 49 +/- 6 kJ/mol, but the enthalpy change is small, -4 +/- 10 kJ/mol. On the time scale we studied, the primary photochemical kinetics, enthalpy, and volume changes are not affected by substituting the solvent D2O for H2O. Comparing data on monomeric and aggregated BR, we conclude that the functional unit for the photocycle is the BR monomer, because both the kinetics (rate constant and activation energy) and the enthalpy changes are independent of its aggregation state.     

Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation by static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane

The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with [4-13C]aspartic acid. An incorporation of ca. 70% was obtained. The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate. Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.     

Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide

Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported [Renthal, R., Dawson, N., & Villarreal, L. (1981) Biochem. Biophys. Res. Commun. 101, 653-657]. We have now located the DCCD site in the bacteriorhodopsin amino acid sequence. [14C]DCCD-bacteriorhodopsin (0.67 mol/mol of bacteriorhodopsin) was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major 14C peptide (50%) and two minor fractions were obtained. The modified peptides were completely absent in the absence of DCCD, and 10 times less was obtained when the reaction was run in the dark. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. Quantitative sequence analysis ruled out significant amounts of DCCD at Glu-74, Asp-85, Asp-96, Asp-102, and Asp-104. The major 14C peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, we conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.     

Perturbation of the tRNA tertiary core differentially affects specific steps of the elongation cycle

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.     

Functional roles of aspartic acid residues at the cytoplasmic surface of bacteriorhodopsin.

The functions of the four aspartic acid residues in interhelical loops at the cytoplasmic surface of bacteriorhodopsin, Asp-36, Asp-38, Asp-102, and Asp-104, were investigated by studying single and multiple aspartic acid to asparagine mutants. The same mutants were examined also with the additional D96N residue replacement. The kinetics of the M and N intermediates of the photochemical cycles of these recombinant proteins were affected only in a minor, although self-consistent, way. When residue 38 is an aspartate and anionic, it makes the internal proton exchange between the retinal Schiff base and Asp-96 about 3 times more rapid, and events associated with the reisomerization of retinal to all-trans about 3 times slower. Asp-36 has the opposite effect on these processes, but to a smaller extent. Asp-102 and Asp-104 have even less or none of these effects. Of the four aspartates, only Asp-36 could play a direct role in proton uptake at the cytoplasmic surface. In the 13 bacterioopsin sequences now available, only this surface aspartate is conserved.     

Protein conformational changes in the bacteriorhodopsin photocycle

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation.Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.     

Structure-function studies on bacteriorhodopsin. VIII. Substitutions of the membrane-embedded prolines 50, 91, and 186: the effects are determined by the substituting amino acids

To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded alpha-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50----Ala, Pro-91----Ala and ----Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50----Gly was 10-fold faster than in the wild-type. While, Pro-186----Ala regenerated the wild-type chromophore, the mutants Pro-186----Val and Pro-186----Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186----Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentration restored the wild-type purple chromophore in the Pro-186----Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186----Ala and Pro-186----Gly showed about 65%, whereas Pro-186----Val showed 10-20% of the normal proton pumping.     

Structural changes in the L photointermediate of bacteriorhodopsin

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.