Influence of the charge at D85 on the initial steps in the photocycle of bacteriorhodopsin.

Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.

From shell to cell: neutron scattering studies of biological water dynamics and coupling to activity.

An integrated picture of hydration shell dynamics and of its coupling to functional macromolecular motions is proposed from studies on a soluble protein, on a membrane protein in its natural lipid environment, and on the intracellular environment in bacteria and red blood cells. Water dynamics in multimolar salt solutions was also examined, in the context of the very slow water component previously discovered in the cytoplasm of extreme halophilic archaea. The data were obtained from neutron scattering by using deuterium labelling to focus on the dynamics of different parts of the complex systems examined.

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.

Evolution in the laboratory: the genome of Halobacterium salinarum strain R1 compared to that of strain NRC-1.

We report the sequence of the Halobacterium salinarum strain R1 chromosome and its four megaplasmids. Our set of protein-coding genes is supported by extensive proteomic and sequence homology data. The structures of the plasmids, which show three large-scale duplications (adding up to 100 kb), were unequivocally confirmed by cosmid analysis. The chromosome of strain R1 is completely colinear and virtually identical to that of strain NRC-1. Correlation of the plasmid sequences revealed 210 kb of sequence that occurs only in strain R1. The remaining 350 kb shows virtual sequence identity in the two strains. Nevertheless, the number and overall structure of the plasmids are largely incompatible. Also, 20% of the protein sequences differ despite the near identity at the DNA sequence level. Finally, we report genome-wide mobility data for insertion sequences from which we conclude that strains R1 and NRC-1 originate from the same natural isolate. This exemplifies evolution in the laboratory.

Dynamics of hydration water in deuterated purple membranes explored by neutron scattering.

The function and dynamics of proteins depend on their direct environment, and much evidence has pointed to a strong coupling between water and protein motions. Recently however, neutron scattering measurements on deuterated and natural-abundance purple membrane (PM), hydrated in H2O and D2O, respectively, revealed that membrane and water motions on the ns-ps time scale are not directly coupled below 260 K (Wood et al. in Proc Natl Acad Sci USA 104:18049-18054, 2007). In the initial study, samples with a high level of hydration were measured. Here, we have measured the dynamics of PM and water separately, at a low-hydration level corresponding to the first layer of hydration water only. As in the case of the higher hydration samples previously studied, the dynamics of PM and water display different temperature dependencies, with a transition in the hydration water at 200 K not triggering a transition in the membrane at the same temperature. Furthermore, neutron diffraction experiments were carried out to monitor the lamellar spacing of a flash-cooled deuterated PM stack hydrated in H2O as a function of temperature. At 200 K, a sudden decrease in lamellar spacing indicated the onset of long-range translational water diffusion in the second hydration layer as has already been observed on flash-cooled natural-abundance PM stacks hydrated in D2O (Weik et al. in J Mol Biol 275:632-634, 2005), excluding thus a notable isotope effect. Our results reinforce the notion that membrane-protein dynamics may be less strongly coupled to hydration water motions than the dynamics of soluble proteins.

Coupling of protein and hydration-water dynamics in biological membranes.

The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H(2)O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D(2)O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps-ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures <260 K. Molecular-dynamics simulations of hydrated PM in the temperature range from 100 to 296 K revealed an onset of hydration-water translational diffusion at approximately 200 K, but no transition in the PM at the same temperature. Our results suggest that, in contrast to soluble proteins, the dynamics of the membrane protein is not controlled by that of hydration water at temperatures <260 K. Lipid dynamics may have a stronger impact on membrane protein dynamics than hydration water.

Modelling the CheY(D10K,Yl00W) Halobacterium salinarum mutant: sensitivity analysis allows choice of parameter to be modified in the phototaxis model.

A recent phototaxis model of Halobacterium salinarum composed of the signalling pathway and the switch complex of the motor explained all considered experimental data on spontaneous switching and response time to repellent or attractant light stimuli. However, the model which considers symmetric processes in the clockwise and counter-clockwise rotations of the motor cannot explain the behaviour of a CheY(D10K,Yl00W) mutant which always moves forward and does not respond to light. We show that the introduction of asymmetry in the motor switch model can explain this behaviour. Sensitivity analysis allowed us to choose parameters for which the model is sensitive and whose values we then change in either direction to obtain an asymmetric model. We also demonstrate numerically that at low concentrations of CheYP, the symmetric and asymmetric models behave similarly, but at high concentrations, differences in the clockwise and counter-clockwise modes become apparent. Thus, those experimental data that could previously be explained only by ad hoc assumptions are now obtained 'naturally' from the revised model.

Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX-.

Low-temperature (1.4 K), single-molecule fluorescence-excitation spectra have been recorded for individual reaction center-light-harvesting 1 complexes from Rhodopseudomonas palustris and the PufX(-) strain of Rhodobacter sphaeroides. More than 80% of the complexes from Rb. sphaeroides show only broad absorption bands, whereas nearly all of the complexes from Rps. palustris also have a narrow line at the low-energy end of their spectrum. We describe how the presence of this narrow feature indicates the presence of a gap in the electronic structure of the light-harvesting 1 complex from Rps. palustris, which provides strong support for the physical gap that was previously modeled in its x-ray crystal structure.

From primary photochemistry to biological function in the blue-light photoreceptors PYP and AppA.

To properly respond to changes in fluency conditions, Nature has developed a variety of photosensors that modulate gene expression, enzyme activity and/or motility. Dedicated types have evolved, which can be classified in six families: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins and BLUF-proteins. The photochemistry of the first three families is based on cis/trans isomerization of an ethylene bond. Surprisingly, the latter three all use flavin as their chromophore, but each with very different photochemistry. In this contribution we will discuss the molecular basis of signal generation in a xanthopsin (Photoactive Yellow Protein (PYP) from Halorhodospira halophila), a photoreceptor for negative phototaxis, and in a BLUF protein (AppA from Rhodobacter sphaeroides), a transcriptional anti-repressor. PYP is activated through trans/cis isomerization of the 7,8-vinyl bond of its 4-hydroxycinnamic acid chromophore. This initiates a photocycle with multiple intermediates, like pB, which is formed after intramolecular proton transfer. The negative charge thus formed in the interior of the protein triggers formation of a partially unfolded signaling state. For AppA much less is known about the underlying photochemistry. Available evidence suggests that it is based on a light-induced change in the hydrogen-bonding of its flavin chromophore and/or a change in hydrophobic stacking between the flavin and/or nearby aromatic amino acids like Y 21. A signaling state is formed within microseconds, which recovers with a rate of approximately 10(-3) s(-1). The change in conformation between receptor- and signaling-state in AppA, however, appear to be minute as compared to those in PYP. Here we review the underlying chemistry in the various steps of the photocycle of these two photoreceptor proteins and provide new data on their mechanism and function.

Proton channel hydration and dynamics of a bacteriorhodopsin triple mutant with an M-state-like conformation.

The hydration and dynamics of purple membranes (PM) containing the bacteriorhodopsin (BR) triple mutant D96G/F171C/F219L were investigated by neutron diffraction coupled with H(2)O/D(2)O exchange and by energy-resolved neutron scattering. The mutant, which is active in proton transport (Tittor et al. in J. Mol. Biol. 319:555-565, 2002), has an "open" ground-state structure similar to that of the M intermediate in the photocycle of the wild type (wt) (Subramaniam and Henderson in Nature 406:653-657, 2000). The experiments demonstrated an increased proton channel hydration in the mutant PM compared with wt PM, in both high (86%) and low (57%) relative humidity. We suggest that this is due to the smaller side chains of the mutant residues liberating space for more water molecules in the proton channel, which would then be able to participate in the proton translocation network. PM thermal dynamics has been shown to be very sensitive to membrane hydration (Lehnert et al. in Biophys. J. 75:1945-1952, 1998). The global dynamical behaviour of the mutant PM on the 100-ps time scale, as a function of relative humidity, was found to be identical to that of the wt, showing that the "open" BR structure and additional water molecules in the proton channel do not provide a softer environment enabling increased flexibility.

Ultrafast conformational dynamics in cyclic azobenzene peptides of increased flexibility.

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.

Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes.

Unlike wild-type bacteriorhodopsin (BR), the BR triple mutant D96G/F171C/F219L has been shown to undergo only minor structural rearrangements during its photocycle. Nonetheless, the mutant is capable of transporting protons at a rate of 125(+/-40) H+/BR per minute under light-saturating conditions. Light adaptation of the triple mutant's retinal proceeds in a pH-dependent manner up to a maximum of 63% all-trans. These two findings imply that the transport activity of the triple mutant comprises 66% of the wild-type activity. Time-resolved spectroscopy reveals that the identity and sequence of intermediates in the photocycle of the triple mutant in the all-trans configuration correspond to that of wild-type BR. The only differences relate to a slower rise and decay of the M and O intermediates, and a significant spectral contribution from a 13-cis component. No indication for accumulation of the N intermediate is found under a variety of conditions that normally favor the formation of this species in wild-type BR. The Fourier transform infrared (FTIR) spectrum of the M intermediate in the triple mutant resembles that of wild type. Minor changes in the amide I region during the photocycle suggest that only small movements of the protein backbone occur. Electron microscopy reveals large differences in conformation between the unilluminated state of the mutant protein and wild-type but no light-induced changes in time-resolved measurements. Evidently, proton transport by the triple mutant does not require the major conformational rearrangements that occur on the same time-scale with wild-type. Thus, we conclude that large conformational changes observed in the photocycle of the wild-type and many BR mutants are not a prerequisite for the change in accessibility of the Schiff base nitrogen atom that must occur during vectorial catalysis to allow proton transport.

Kinetics, energetics, and electronic coupling of the primary electron transfer reactions in mutated reaction centers of Blastochloris viridis.

Femtosecond spectroscopy in combination with site-directed mutagenesis has been used to study the dynamics of primary electron transfer in native and 12 mutated reaction centers of Blastochloris (B) (formerly called Rhodopseudomonas) viridis. The decay times of the first excited state P* vary at room temperature between of 0.6 and 50 ps, and at low temperatures between 0.25 and 90 ps. These changes in time constants are discussed within the scope of nonadiabatic electron transfer theory using different models: 1) If the mutation is assumed to predominantly influence the energetics of the primary electron transfer intermediates, the analysis of the room temperature data for the first electron transfer step to the intermediate P(+)B(A)(-) yields a reorganization energy lambda = 600 +/- 200 cm(-1) and a free energy gap Delta G ranging from -600 cm(-1) to 800 cm(-1). However, this analysis fails to describe the temperature dependence of the reaction rates. 2) A more realistic description of the temperature dependence of the primary electron transfer requires different values for the energetics and specific variations of the electronic coupling upon mutation. Apparently the mutations also lead to pronounced changes in the electronic coupling, which may even dominate the change in the reaction rate. One main message of the paper is that a simple relationship between mutation and a change in one reaction parameter cannot be given and that at the very least the electronic coupling is changed upon mutation.

Lag1p and Lac1p are essential for the Acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisae.

Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1 Delta lac1 Delta cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA-independent ceramide synthesis activity. Quadruple mutant cells (lag1 Delta lac1 Delta ypc1 Delta ydc1 Delta) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1 Delta lac1 Delta cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.

The fla gene cluster is involved in the biogenesis of flagella in Halobacterium salinarum.

In this study, a flagella-related protein gene cluster is described for Halobacterium salinarum. The fla gene cluster is located upstream of the flagellin genes flgB1-3 and oriented in the opposite direction. It consists of nine open reading frames (ORFs): htpIX, a member of the halobacterial transducer protein gene family, and the genes flaD-K. The genes flaD, E, G, H, I and J share high homologies with genes from other Archaea. Interestingly, flaK shows similarities to bacterial genes involved in the regulation of flagellar synthesis. The ORFs of flaH, flaI and flaK contain sequences coding for nucleotide binding sites. Furthermore, flaI contains a motif called the bacterial type II secretion protein E signature, indicating a functional relation to members of the bacterial pili type IV-type II secretion protein superfamily. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the genes flaE to flaK are transcribed into one polycistronic message. In frame deletion mutants of flaI were generated by gene replacement. The deletion strain lacks motility and belongs to the fla(-) mutant class, indicating that it is deficient in flagellar biogenesis. The overall amount of flagellin protein in Delta flaI cells is reduced, although transcription of the flagellin genes is unaffected. Therefore, the flaI gene product is involved in the biosynthesis, transport or assembly of flagella in H. salinarum.

Roles of cytoplasmic arginine and threonine in chloride transport by the bacteriorhodopsin mutant D85T.

In the light-driven anion pump halorhodopsin (HR), the residues arginine 200 and threonine 203 are involved in anion release at the cytoplasmic side of the membrane. Because of large sequence homology and great structural similarities between HR and bacteriorhodopsin (BR), it has been suggested that anion translocation by HR and by the chloride-pumping BR mutant BR-D85T occurs by the same mechanism. Consequently, the functions of the R200/T203 pair in HR should be the same as those of the corresponding pair in BR-D85T (R175/T178). We have put this hypothesis to a test by creating two mutants of BR-D85T in which R175 and T178 were replaced by glutamine and valine, respectively. Chloride transport activities were essentially the same for all three mutants, whereas chloride binding and the kinetics of parts of the photocycle were markedly affected by the replacement of T178. In contrast, the consequences of mutating R175 proved to be less significant. These findings are consistent with evidence obtained on HR and therefore support the idea that the respective mechanistic roles of the cytoplasmic arginine/threonine pairs in HR and BR-D85T are equal.

Dynamics of the proton transfer reaction on the cytoplasmic surface of bacteriorhodopsin.

The cytoplasmic surface of bacteriorhodopsin is characterized by a group of carboxylates that function as a proton attractive domain [Checover, S., Nachliel, E., Dencher, N. A., and Gutman, M. (1997) Biochemistry 36, 13919-13928]. To identify these carboxylates, we selectively mutated them into cysteine residues and monitored the effects of the dynamics of proton transfer between the bulk and the surface of the protein. The measurements were carried out without attachment of a pH-sensor to the cysteine residue, thus avoiding any structural perturbation and change in the surface charge caused by the attachment of a reporter group, and the protein was in its BR state. The purple membranes were suspended in an unbuffered solution of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) and exposed to a train of 1000 laser pulses (2.1 mJ/pulse, lambda = 355 nm, at 10 Hz). The excitation of the dye ejected the hydroxyl's proton, and a few nanoseconds later, a pair of free protons and ground-state pyranine anion was formed. The experimental observation was the dynamics of the relaxation of the system to the prepulse state. The observed signals were reconstructed by a numeric method that replicates the chemical reactions proceeding in the perturbed space. The detailed reconstruction of the measured signal assigned the various proton-binding sites with rate constants for proton binding and proton exchange and the pK values. Comparison of the results obtained by the various mutants indicates that the dominant proton-binding cluster of the wild-type protein consists of D104, E161, and E234. The replacement of D104 or E161 with cysteine lowered the proton binding capacity of the cluster to approximately 60% of that of the native protein. The replacement of E234 with cysteine disrupted the structure of the cluster, causing the two remaining carboxylates to function as isolated residues that do not interact with each other. The possibility of proton transfer between monomers is discussed.

Primary reactions of sensory rhodopsins.

The first steps in the photocycles of the archaeal photoreceptor proteins sensory rhodopsin (SR) I and II from Halobacterium salinarum and SRII from Natronobacterium pharaonis have been studied by ultrafast pump/probe spectroscopy and steady-state fluorescence spectroscopy. The data for both species of the blue-light receptor SRII suggests that their primary reactions are nearly analogous with a fast decay of the excited electronic state in 300-400 fs and a transition between two red-shifted product states in 4-5 ps. Thus SRII behaves similarly to bacteriorhodopsin. In contrast for SRI at pH 6.0, which absorbs in the orange part of the spectrum, a strongly increased fluorescence quantum yield and a drastically slower and biexponential decay of the excited electronic state occurring on the picosecond time scale (5 ps and 33 ps) is observed. The results suggest that the primary reactions are controlled by the charge distribution in the vicinity of the Schiff base and demonstrate that there is no direct connection between absorption properties and reaction dynamics for the retinal protein family.

Photoresponsive dendritic azobenzene peptides.

Two dendritic peptides containing a branched lysine core and up to eight azobenzene moieties in the periphery were synthesized on solid support employing the omega-amino acid 4-(aminomethyl)phenylazobenzoic acid. With an additional peptidic tail consisting of an oligolysine portion, water solubility was achieved for the dendrimers, which allowed for the characterization of the cis/trans photoisomerization of the dendritic azobenzene species in both organic and aqueous media. Despite the interactions between the chromophores, which occur particularly in aqueous media, at higher dilution the photoisomerization process was found to proceed to extents that should permit photomodulation of molecular recognition processes between ligands grafted to the photosensitive azobenzene units and receptor molecules.

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Balstochloris viridis. Influence of mutations at position M208.

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (phi(*-)A) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BCh1), B(A), and the BPh, phiA, that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of (phi(*-)A at 160 K indicates that two distinct states of phi(*-)A are present in the wild type and the mutant YF(M208). Based on a comparison with phi(*-)A in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of phiA. Only one phi(*-)A state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state phi(*-)AQ(*-)A is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294-302]. The corresponding absorbance changes in the BCh1 Q(x) and Q(y) regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of phiA changes its orientation. It is concluded that this distinct structural relaxation of phiA can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.