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PubMed2003

Decisive role of electronic polarization of the protein environment in determining the absorption maximum of halorhodopsin.

It is known that the absorption maximum of halorhodopsin is red shifted by 10 nm with the uptake of a chloride ion Cl(-). According to the X-ray structure, the ion is located at the position of the counterion of the chromophore, protonated retinal Schiff base. Thus, the direction of the observed spectral change is opposite to that expected from the pi-electron redistribution (an increase in the bond alternation) induced by the counterion. The physical origin of this abnormal shift is never explained in terms of any simple chemical analogues. We successfully explain this phenomenon by a QM/MM type of excitation energy calculation. The three-dimensional structure of the protein is explicitly taken into account using the X-ray structure. We reveal that the electronic polarization of the protein environment plays an essential role in tuning the absorption maximum of halorhodopsin.

Chlorides↗
PubMed2003

Occupancy of two primary chloride-binding sites in Natronobacterium pharaonis halorhodopsin is a necessary condition for active anion transport.

The existence of two primary chloride-binding sites was found on the basis of the study of halorhodopsin spectra at different chloride concentrations. SVD analysis of the spectra revealed two chloride-dependent components at low chloride concentration (0.1-10 mM). Global fitting of SVD components found K(D) values of 0.47 and 5.2 mM with unitity Hill coefficients. The second K(D) coincides with the apparent K(D) of the photovoltage response of halorhodopsin.

Binding Sites↗
PubMed2000

The three-dimensional structure of halorhodopsin to 5 A by electron crystallography: A new unbending procedure for two-dimensional crystals by using a global reference structure.

Electron microscopy does not, in principle, require highly ordered crystals to determine a high-resolution structure. Nevertheless, crystals of any type help to constrain the molecules into a more limited range of orientations and positions, from which it is easier to carry out structure determination. We describe an improved procedure for determination of crystalline disorder, which we have applied to poorly ordered two-dimensional crystals of the chloride pump halorhodopsin from Halobacterium salinarum. The new image analysis procedure involves the use of a reference projection calculated from a global three-dimensional map to carry out the initial cross-correlation analysis. Coupled with a greater number of images taken with field emission gun microscopes, this has allowed us to calculate a three-dimensional structure for halorhodopsin, in which the seven transmembrane helices and certain molecular features, such as the beta-ionone ring of retinal, are now resolved.

Bacteriorhodopsins↗
PubMedDate not supplied

The crystal structure of the L1 intermediate of halorhodopsin at 1.9 angstroms resolution.

The mutant T203V of the light driven chloride pump halorhodopsin from Halobacterium salinarum was crystallized and the X-ray structure was solved at 1.6 angstroms resolution. The T203V structure turned out to be nearly identical to the wild type protein with a root mean square deviation of 0.43 angstroms for the carbon alpha atoms of the protein backbone. Two chloride binding (CB) sites were demonstrated by a substitution of chloride with bromide and an analysis of anomalous difference Fourier maps. The CB1 site was found at the same position as in the wild type structure. In addition, a second chloride binding site CB2 was identified around Q105 due to higher resolution in the mutant crystal. As T203V showed a 10 times slower decay of its photocycle intermediate L, this intermediate could be trapped with an occupancy of 60% upon illumination at room temperature and subsequent cooling to 120 degrees K. Fourier transform infrared spectroscopy clearly identified the crystal to be trapped in the L1 intermediate state and the X-ray structure was solved to 1.9 angstroms resolution. In this intermediate, the chloride moved by 0.3 angstroms within binding site CB1 as indicated by peaks in difference Fourier density maps. The chloride in the second binding site CB2 remained unchanged. Thus, intraproteinous chloride translocation from the extracellular to the cytoplasmic part of the protein must occur in reaction steps following the L1 intermediate in the catalytic cycle of halorhodopsin.

Amino Acid Substitution↗
PubMed1998

Photophosphorylation in alkalophilic halobacterial cells containing halorhodopsin: chloride-ion cycle?

Light-driven ATP synthesis is found in cells of the alkalophilic bacterium Natronobacterium pharaonis containing halorhodopsin but deficient in H+-pumping bacteriorhodopsin. Photophosphorylation occurs with cyanide-inhibited respiratory chain as well as without cyanide in conditions with low C1- concentration in the incubation medium. Increase in C1- concentration from 0.1 to 2.35 M in the incubation medium leads to inhibition of photophosphorylation. Continuous illumination increases membrane Delta Psi if respiration is inhibited by cyanide. This effect is stimulated by DCCD, an ATPase inhibitor. These data can be explained if one suggests that halorhodopsin pumps C1- into the cells whereas C1- release from the cells through C1--ATP-synthase is coupled with the ATP synthesis (chloride-ion cycle).

Adenosine Triphosphate↗
PubMed1996

Modeling of halorhodopsin and rhodopsin based on bacteriorhodopsin.

Bacteriorhodopsin (BR), halorhodopsin (HR), and rhodopsin (Rh) all belong to the class of seven-helix membrane proteins. For BR, a structural model at atomic resolution is available; for HR, diffraction data are available only down to a resolution of 6 A in the membrane plane, and for Rh, down to 9 A. BR and HR are closely related proteins with a sequence homology of 34%, while Rh does not share any sequence homology with BR. An atomic model for HR is derived that is based on sequence alignment and the atomic model for BR and is improved by molecular dynamics simulations. The model structure obtained accounts well for the experimentally observed difference between HR and BR in the projection map, where HR exhibits a higher density in the region between helices D and E. The reason for this difference lies partially in the different side chains and partially in slightly different helix tilts. The scattering amplitudes and phases of the model structure are calculated and agree with the experimental data down to a resolution of about 8 A. If the helix positions are adopted from the projection map for HR and used as input in the model, this number improves to 7 A. Analogously, an atomic model for Rh is derived based on the atomic model for BR and subjected to molecular dynamics simulations. Optimal agreement with the experimental projection map for Rh is obtained when the entire model structure is rotated slightly about two axes in the membrane plane. The agreement with the experimental projection map is not as satisfactory as for HR, but the results indicate that even for a nonhomologous, but structurally related, protein such as Rh, an acceptable model structure can be derived from the structure of BR.

Bacteriorhodopsins↗
PubMed1983

Isolation and properties of the native chromoprotein halorhodopsin.

The native chromoprotein of the light-driven chloride pump halorhodopsin (HR) was isolated from Halobacterium halobium strain L-33 which lacks bacteriorhodopsin but contains 'slow cycling rhodopsin-like pigment' (SR). A membrane fraction was prepared in low salt and dissolved in a high salt medium by the detergents Lubrol PX or octylglucoside. These conditions destroyed the chromophore of SR but not the HR pigment. Chromatography on phenyl-Sepharose and hydroxylapatite produced, in 60% yield, a 230-fold enriched monomeric chromoprotein with an apparent mol. wt. of 20,000. The chromoprotein was stable in 1 M NaCl and 1% octylglucoside and remained stable upon removal of detergent. It reacted with borohydride in the dark and with hydroxylamine in the light. The absorption maximum of the light-adapted state is at 580 + 2 nm and its molar extinction approximately 50,000/M/cm. Upon illumination in the presence of detergent it was converted into a 410 nm absorbing species with concomitant release of protons. A thermal reconversion to the 580 nm species occurred with a half time of 76 s at -6 degrees C. Blue light absorbed by the photoproduct accelerated the re-conversion as well as the re-uptake of protons. Removal of the detergent prevented the light-induced formation of the 410 nm species. Under these conditions a photochemical behaviour similar to that in intact cells and cell vesicles, i.e., a photocycle in the 10-20 ms range was observed. These findings form the basis for functional reconstitution of HR.

Bacteriorhodopsins↗
PubMed1985

The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump.

Halorhodopsin, the light-driven chloride pump of halobacteria, undergoes a photochemical cycle in the 10 ms range. Two intermediates, HR640 and HR520, accumulate in the photosteady state after short times (within 100 ms) of illumination. Upon prolonged illumination a third species, HRL410 accumulates, which is formed from HR520/HR640 by deprotonation of the chromophore in a side reaction of the photocycle. In the dark, HRL410 requires several minutes to reconvert thermally to HR478. Thus, molecules in the HRL410 state must be inactive pumps since their maximal turnover number could only be a few per hour. Inorganic bases, such as azide, catalyze the deprotonation of HR520/HR640 as well as the reprotonation of HRL410. Both reactions are accelerated several hundred times by azide but the photosteady-state concentration of HRL410 remains unchanged.

Azides↗
PubMed1987

The halo-opsin gene. II. Sequence, primary structure of halorhodopsin and comparison with bacteriorhodopsin.

The gene for the protein moiety of the light-driven chloride pump halorhodopsin (HR), hop gene, was sequenced and the primary structure of the protein derived thereof. The gene has a GC content of 67% and codes for 274 amino acids. A promoter structure, resembling that of the halobacterial 16S rRNA genes, is present and both a terminating stem and a loop sequence is found downstream of the TGA stop codon. A ribosomal binding site is located within the translated region. The HR protein moiety is processed at the amino terminus, as well as the carboxy terminus, yielding a dominant species of calculated Mr 26 961.

Amino Acid Sequence↗
PubMed1988

Structure and orientation of halorhodopsin in the membrane: a proteolytic fragmentation study.

Halorhodopsin (HR), the light-driven chloride pump in halobacteria, was digested with various proteolytic enzymes. As expected, carboxypeptidase A removed 14 amino acids from the C-terminal tail of detergent-solubilized HR, producing a fragment of 25.2 kd in size. Membrane-associated HR could be digested as well, but not in right-side-out sealed cell envelope vesicles. We conclude, therefore, that the orientation of HR in the cytoplasmic membrane is such that the C-terminal tail faces the cytoplasmic side. Tryptic digestion of detergent-solubilized HR resulted in the removal of the same C-terminal segment, but also in the production of two more cleavage products (molecular masses of 20.9 and 16.8 kd respectively). These cleavage sites were determined by amino acid sequencing of the newly produced N termini, and they turned out to be within interhelical loops in an earlier proposed structural model for HR. Incubation with chymotrypsin and thermolysin yielded different sites of cleavage, but also in regions which were proposed to be accessible on the surface of the protein. Since the results show that three of six proposed interhelical loop segments contain proteolytic digestion sites, they support the proposed structural model for HR.

Amino Acid Sequence↗
PubMed1983

Light-induced delta pH of envelope vesicles containing halorhodopsin measured by use of a spin probe.

The spin-labeled amine, 4-amino-2,2,6,6-tetramethyl-piperidino-N-oxyl was used to measure the photoinduced delta pH in the envelope vesicles derived from mutant cells of Halobacterium halobium. The cells contain halorhodopsin (hR) and are spectroscopically free from bacteriorhodopsin (bR). The EPR signals from the extravesicular populations of the probe was quenched by ferricyanide. The membrane bound population of the probe was less than 1% of the amount of the probe entrapped within the vesicle. Hence, we can monitor continuously the concentration of the intravesicular populations of the probe and delta pH can be calculated from the line height of the signal. The kinetic analysis indicated that the time course of the change in the EPR signal represents faithfully delta pH of the vesicle. At pH 7.5, the photoinduced delta pH showed a maximum. The kinetic constant of pH change also displayed a peak at this pH. Addition of uncoupler did not increase either the rate nor magnitude of delta pH at this pH, but at other pH, especially at lower pH, uncoupler showed its effect. A theoretical equation was derived which correlated the photocycle of hR with delta pH. According to the analysis by use of this equation, the time constant of the photocycle was suggested to be constant in pH ranging from 5.5 to 8.25. The maximum in photoinduced delta pH observed at pH 7.5 was concluded to be due to the permeability dependence of ions, especially of H+ on pH. The temperature dependence of delta pH was measured. As the temperature decreased, delta pH was, surprisingly, found to be increased. The rate constant of the photocycle analyzed by the equation, however, decreased with decrease of temperature.

Bacterial Proteins↗
PubMed1995

Over-expression of a new photo-active halorhodopsin in Halobacterium salinarium.

The gene of haloopsin (hop) from halobacterial strain shark was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of shark halorhodopsin (HR) showed that its homology with halobium HR was 62%. The gene product seems to be HR having several positively charged residues that are conserved in all known HRs. The gene encoding shark hop as well as that encoding halobium hop were successfully expressed in Halobacterium salinarium (halobium) by using a plasmid shuttle vector containing the bacterioopsin (bop) promoter. The expression level of shark HR is almost the same as that for halobium HR with the same shuttle vector containing the bop promoter. Under the physiological conditions, the anion pumping activity of the shark HR expressed in H. salinarium was almost the same as that for halobium HR; however, the anion selectivity and half-maximal anion transport were different. Furthermore, its absorption maximum in the absence of chloride shifted to approx. 596 nm in contrast to that for halobium HR. The half-lifetimes of HR520 formation for shark HR and halobium HR were almost the same; however, the half-lifetime of its decay was approx. 6-times faster for shark HR than it was for halobium HR at a high chloride concentration (1000 mM). Even at a low chloride concentration (50 mM), HR520 and HR640 intermediates could be detected for shark HR, and the half-lifetime of HR640 decay was found to be approx. 25 ms. In the presence of nitrate, the half-lifetime of HR565 recovery for shark HR was approx. 10-times slower than that for halobium HR. Some of amino acid substitutions between shark HR and halobium HR may affect the anion selectivity and the photoreaction of HR.

Amino Acid Sequence↗
PubMed1987

The leader peptides from bacteriorhodopsin and halorhodopsin are potential membrane-spanning amphipathic helices.

We show that the N-terminal leader peptides from the bacterial membrane proteins bacteriorhodopsin and halorhodopsin can be expected to form amphipathic alpha-helics with a highly hydrophobic nonpolar face and a narrow, negatively charged polar face. This finding is discussed in terms of a model for the integration of these proteins into the bacterial membrane.

Bacteriorhodopsins↗
PubMedDate not supplied

Differences between the photocycles of halorhodopsin and the acid purple form of bacteriorhodopsin analyzed with millisecond time-resolved FTIR spectroscopy.

At pH 1, bacteriorhodopsin (bR) is thought to function as a halide ion pump, in contrast to its biological function as a proton pump at neutral pH. Despite the apparent similarity in function between this 'acid purple' form of bR and the native form of halorhodopsin (hR), their FTIR difference spectra measured ca. 5 ms after photolysis are significantly different. The most striking difference is the appearance of a positive band at 1753 cm-1 and a negative band at 1732 cm-1 in the bRacid purple difference spectrum. These and other spectral features are similar, but not identical, to those of the bR-->O difference spectrum measured at neutral pH. The structure of the bRacid purple longest-lived product therefore corresponds more closely to the O photoproduct of the bR proton-pumping photocycle, rather than the hL photoproduct seen on a similar time scale in the hR photocycle. The 1753- and 1732-cm-1 bands are largely unaffected by the D212N mutation, but both appear to lose a portion of their intensities with either the D85N or D96N mutation. Thus Asp-85 and -96 likely undergo substantial changes in hydrogen-bonding environment during the halide-pumping cycle of bRacid purple. Our FTIR results deepen the distinctions between the hR and bR photocycles. The mechanism of chloride pumping in hR has been thought not to involve protonation or hydrogen bonding changes of carboxylic acid groups. In bRacid purple, however, it seems likely that at least one carboxylic acid might play an important role in the mechanism of chloride pumping, leading to an increase in thermodynamic or kinetic stabilization of the O intermediate.

Bacteriorhodopsins↗
PubMed2005

Crystallization of halorhodopsin from Halobacterium sp. shark.

The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.

Crystallization↗
PubMed2005

The influence of the halide ions on the photochemical reaction cycle of pharaonis halorhodopsin.

Sodium salt of chloride, bromide and iodide were used to elucidate the effect of the size of the anion on the binding to pharaonis halorhodopsin and its transport during the photocycle of this retinal protein. Spectroscopic titration revealed an apparent strong binding constant of 2 mM for chloride, 0.23 mM for bromide and 5 mM for iodide. In the case of iodide a second, week binding constant of about 10 M could be estimated. This second binding constant was similar to that observed earlier for nitrate. By changing the halide ions, only the transitions in the second half of the photocycle were affected, which contained intermediates N, O, and HR'. The O to HR' transition becomes faster with increasing ion volume, meaning that the ion uptake is accelerated. This effect shows a direct correlation with the ion radius. With increasing ion concentration the N-O-HR' equilibrium changed in such a way that the accumulated O tended to decrease. This tendency was overruled in iodide, by the appearance of the second binding constant. The increasing iodide concentration, up to 100mM decreases the accumulation of the intermediate O, due to kinetic reasons, but at higher ion concentration the amount of O increases, although its decay becomes faster. This effect correlates with the appearance of the second iodide bound to the protein.

Binding Sites↗
PubMed2005

Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin.

Single-molecule atomic force microscopy and spectroscopy were applied to detect molecular interactions stabilizing the structure of halorhodopsin (HR), a light-driven chloride pump from Halobacterium salinarum. Because of the high structural and sequence similarities between HR and bacteriorhodopsin, we compared their unfolding pathways and polypeptide regions that established structurally stable segments against unfolding. Unfolding pathways and structural segments stabilizing the proteins both exhibited a remarkably high similarity. This suggests that different amino acid compositions can establish structurally indistinguishable energetic barriers. These stabilizing domains rather result from comprehensive interactions of all amino acids within a structural region than from specific interactions. However, one additional unfolding barrier located within a short segment of helix E was detected for HR. This barrier correlated with a Pi-bulk interaction, which locally disrupts helix E and divides a structural stabilizing segment.

Amino Acid Sequence↗
PubMed2005

Mechanism of a molecular valve in the halorhodopsin chloride pump.

Halorhodopsin is a light-driven chloride anion pump in which the trans-->cis photoisomerization of a retinal chromophore triggers a photocycle resulting in the translocation of chloride across the plasma membrane. The mechanism of chloride transfer past the cis retinal is determined here by computing multiple pathways for this process. The calculations reveal two conditions of the valve mechanism. First, a lumen absent in the ground state structure is transiently opened by chloride passage. Second, this activated opening, which is achieved by flexible deformation of the surrounding protein, is shown to significantly raise the chloride translocation barrier between photocycles, thus preventing chloride backflow. Unlike macroscopic valve designs, the protein allows differential ion flows in the pumping and resting states that are tuned to match the physiological timescales of the cell, thus creating a "kinetic" valve.

Chlorides↗