Location of a cation-binding site in the loop between helices F and G of bacteriorhodopsin as studied by 13C NMR. (1/379)

The high-affinity cation-binding sites of bacteriorhodopsin (bR) were examined by solid-state 13C NMR of samples labeled with [3-13C]Ala and [1-13C]Val. We found that the 13C NMR spectra of two kinds of blue membranes, deionized (pH 4) and acid blue at pH 1.2, were very similar and different from that of the native purple membrane. This suggested that when the surface pH is lowered, either by removal of cations or by lowering the bulk pH, substantial change is induced in the secondary structure of the protein. Partial replacement of the bound cations with Na+, Ca2+, or Mn2+ produced additional spectral changes in the 13C NMR spectra. The following conclusions were made. First, there are high-affinity cation-binding sites in both the extracellular and the cytoplasmic regions, presumably near the surface, and one of the preferred cation-binding sites is located at the loop between the helix F and G (F-G loop) near Ala196, consistent with the 3D structure of bR from x-ray diffraction and cryoelectron microscopy. Second, the bound cations undergo rather rapid exchange (with a lifetime shorter than 3 ms) among various types of cation-binding sites. As expected from the location of one of the binding sites, cation binding induced conformational alteration of the F-G interhelical loop.  (+info)

Car: a cytoplasmic sensor responsible for arginine chemotaxis in the archaeon Halobacterium salinarum. (2/379)

A new metabolic signaling pathway for arginine, both a chemoeffector and a fermentative energy source, is described for Halobacterium salinarum. Systematic screening of 80+ potentially chemotactic compounds with two behavioral assays identified leucine, isoleucine, valine, methionine, cysteine, arginine and several peptides as strong chemoattractants. Deletion analysis of a number of potential halobacterial transducer genes led to the identification of Car, a specific cytoplasmic arginine transducer which lacks transmembrane helices and was biochemically shown to be localized in the cytoplasm. Flow assays were used to show specific adaptive responses to arginine and ornithine in wild-type but not Deltacar cells, demonstrating the role of Car in sensing arginine. The signaling pathway from external arginine to the flagellar motor of the cell involves an arginine:ornithine antiporter which was quantitatively characterized for its transport kinetics and inhibitors. By compiling the chemotactic behavior, the adaptive responses and the characteristics of the arginine:ornithine antiporter to arginine and its analogs, we now understand how the combination of arginine uptake and its metabolic conversion is required to build an effective sensing system. In both bacteria and the archaea this is the first chemoeffector molecule of a soluble methylatable transducer to be identified.  (+info)

Simulation analysis of the retinal conformational equilibrium in dark-adapted bacteriorhodopsin. (3/379)

In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two conformers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two forms, using molecular mechanics and dynamics calculations. Adiabatic potential energy mapping indicates that whereas the twofold intrinsic torsional potentials of the C13==C14 and C15==N16 double bonds favor a sequential torsional pathway, the protein environment favors a concerted, bicycle-pedal mechanism. Which of these two pathways will actually occur in bR depends on the as yet unknown relative weight of the intrinsic and environmental effects. The free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type protein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within approximately kBT of experiment. In contrast, in isolated retinal the free energy of the all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. The free energy differences are similar to the adiabatic potential energy differences in the various systems examined, consistent with an essentially enthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein-protein interactions. Removing internal water molecules near the Schiff base strongly stabilizes the (13,15)cis form, whereas a double mutation that removes negative charges in the retinal pocket (Asp85 to Ala; Asp212 to Ala) has the opposite effect.  (+info)

Chloride ion binding to bacteriorhodopsin at low pH: an infrared spectroscopic study. (4/379)

Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-induced ion pumps in the cell membrane of Halobacterium salinarium. Under normal conditions bR is an outward proton transporter, whereas hR is an inward Cl- transporter. There is strong evidence that at very low pH and in the presence of Cl-, bR transports Cl- ions into the cell, similarly to hR. The chloride pumping activity of bR is connected to the so-called acid purple state. To account for the observed effects in bR a tentative complex counterion was suggested for the protonated Schiff base of the retinal chromophore. It would consist of three charged residues: Asp-85, Asp-212, and Arg-82. This quadruplet (including the Schiff base) would also serve as a Cl- binding site at low pH. We used Fourier transform infrared difference spectroscopy to study the structural changes during the transitions between the normal, acid blue, and acid purple states. Asp-85 and Asp-212 were shown to participate in the transitions. During the normal-to-acid blue transition, Asp-85 protonates. When the pH is further lowered in the presence of Cl-, Cl- binds and Asp-212 also protonates. The binding of Cl- and the protonation of Asp-212 occur simultaneously, but take place only when Asp-85 is already protonated. It is suggested that HCl is taken up in undissociated form in exchange for a neutral water molecule.  (+info)

Time-resolved absorption and photothermal measurements with sensory rhodopsin I from Halobacterium salinarum. (5/379)

An expansion accompanying the formation of the first intermediate in the photocycle of transducer-free sensory rhodopsin I (SRI) was determined by means of time-resolved laser-induced optoacoustic spectroscopy. For the native protein (SRI-WT), the absolute value of the expansion is approximately 5.5 mL and for the mutant SRI-D76N, approximately 1.5 mL per mol of phototransformed species (in 0.5 M NaCl), calculated by using the formation quantum yield for the first intermediate (S610) of Phi610 = 0.4 +/- 0.05 for SRI-WT and 0.5 +/- 0.05 for SRI-D76N, measured by laser-induced optoacoustic spectroscopy and by laser flash photolysis. The similarity in Phi610 and in the determined value of the energy level of S610, E610 = (142 +/- 12) kJ/mol for SRI-WT and SRI-D76N indicates that Asp76 is not directly involved in the first step of the phototransformation. The increase with pH of the magnitude of the structural volume change for the formation of S610 in SRI-WT and in SRI-D76N upon excitation with 580 nm indicates also that amino acids other than Asp76, and other than those related to the Schiff base, are involved in the process. The difference in structural volume changes as well as differences in the activation parameters for the S610 decay should be attributed to differences in the rigidity of the cavity surrounding the chromophore. Except for the decay of the first intermediate, which is faster than in the SRI-transducer complex, the rate constants of the photocycle for transducer-free SRI in detergent suspension are strongly retarded with respect to wild-type membranes (this comparison should be done with great care because the preparation of both samples is very different).  (+info)

Halobacterial rhodopsins. (6/379)

Following the discovery of the bacteriorhodopsin proton pump in Halobacterium halobium (salinarum), not only the halorhodopsin halide pump and two photosensor rhodopsins (sensory rhodopsin and phoborhodopsin) in the same species, but also homologs of these four rhodopsins in strains of other genera of Halobacteriaceae have been reported. Twenty-eight full (and partial) sequences of the genomic DNA of these rhodopsins have been analyzed. The deduced amino acid sequences have led to new strategies and tactics for understanding bacterial rhodopsins on a comparative basis, as summarized briefly in this article. The data discussed include (i) alignment of the sequences to qualify/characterize the conserved residues; (ii) assignment of residues that cause differences in function(s)/properties; and (iii) phylogeny of the halobacterial rhodopsins to suggest their evolutionary paths. The four kinds of rhodopsin in each strain are assumed, on the basis of their genera-specific distributions, to have arisen by at least two gene-duplication processes during evolution prior to generic speciation. The first duplication of the rhodopsin ancestor gene yielded two genes, each of which was duplicated again to give four genes in the ancestor halobacterium. The bacterium carrying four rhodopsin genes, after accumulating mutations, became ready for generic speciation and the delivery of four rhodopsins to each species. The original rhodopsin ancestor is speculated to be closest to the proton pump (bacteriorhodopsin).  (+info)

Protective roles of bacterioruberin and intracellular KCl in the resistance of Halobacterium salinarium against DNA-damaging agents. (7/379)

Halobacterium salinarium, a member of the extremely halophilic archaebacteria, contains a C50-carotenoid namely bacterioruberin. We have previously reported the high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV). In this study, we have examined whether bacterioruberin and the highly concentrated salts in this bacterium play protective roles against the lethal actions of ionizing radiation, UV, hydrogen peroxide, and mitomycin-C (MMC). The colourless mutant of H. salinarium deficient in bacterioruberin was more sensitive than the red-pigmented wild-type to all tested DNA-damaging agents except MMC. Circular dichroism (CD) spectra of H. salinarium chromosomal DNA at various concentrations of KCl (0-3.5 M) were similar to that of B-DNA, indicating that no conformational changes occurred as a result of high salt concentrations. However, DNA strand-breaks induced by ionizing radiation were significantly reduced by the presence of either bacterioruberin or concentrated KCl, presumably due to scavenging of free radicals. These results suggest that bacterioruberin and intracellular KCl of H. salinarium protect this organism against the lethal effects of oxidative DNA-damaging agents.  (+info)

Intermediate spectra and photocycle kinetics of the Asp96 --> asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling. (8/379)

Singular value decomposition with self-modeling is applied to resolve the intermediate spectra and kinetics of the Asp96 --> Asn mutant bacteriorhodopsin. The search for the difference spectra of the intermediates is performed in eigenvector space on the stoichiometric plane. The analysis of data at pH values ranging from 4 to 8 and temperatures between 5 and 25 degrees C reveals significant, early partial recovery of the initial state after photoexcitation. The derived spectra are not biased by assumed photocycles. The intermediate spectra derived in the initial step differ from spectra determined in prior analyses, which results in intermediate concentrations with improved stoichiometric properties. Increasingly more accurate photocycles follow with increasing assumed complexity, of which parallel models are favored, consistent with recent, independent experimental evidence.  (+info)