Janos Lanyi
University of California, Irvine, Physiology and Biophysics, Faculty Member
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Retinal proteins function as photoreceptors and ion pumps. Xanthorhodopsin of Salinibacter ruber is a recent addition to this diverse family. Its novel and distinctive feature is a second chromophore, a caro- tenoid, which serves as... more
Retinal proteins function as photoreceptors and ion pumps. Xanthorhodopsin of Salinibacter ruber is a recent addition to this diverse family. Its novel and distinctive feature is a second chromophore, a caro- tenoid, which serves as light-harvesting antenna. Here we discuss the properties of this carotenoid/retinal complex most relevant to its function (such as the specific binding site controlled by the retinal) and its relationship to other retinal proteins (bacteriorho- dopsin, archaerhodopsin, proteorhodopsin and retinal photoreceptors of archaea and eukaryotes). Antenna addition to a retinal protein has not been observed among the archaea and emerged in bacteria appa- rently in response to environmental conditions where light-harvesting becomes a limiting factor in retinal protein functioning.
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In the last grant period we explored the Na+ binding site of the recently discovered light-driven sodium ion pump. The rationale was that comparison of this novel system to the similar proton pumps and chloride ion pumps would reveal the... more
In the last grant period we explored the Na+ binding site of the recently discovered light-driven sodium ion pump. The rationale was that comparison of this novel system to the similar proton pumps and chloride ion pumps would reveal the amazingly (and unexpectedly) wide variety of structural features that govern conversion of light-energy into biologically useful transmembrane gradients and thus production of biomass. A thorough description of this system would establish the basis for continuing our funded research on these proteins.
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A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion... more
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding ...
Research Interests: Biochemistry, Biophysics, Chemistry, Kinetics, D-Aspartic Acid, and 13 moreSchiff bases, Sodium, Fourier transform infrared spectroscopy, Amino Acid Sequence, Amino Acid Substitution Rates, Recombinant Proteins, Hydrogen-Ion Concentration, Site-directed Mutagenesis, Biochemistry and cell biology, Photochemical Processes, Molecular Sequence Data, binding sites, and Medical biochemistry and metabolomics
The time course of thermal reactions after illumination of 100% humidified bacteriorhodopsin films was followed with FTIR spectroscopy between 125 and 195 K. We monitored the conversion of the initial photoproduct, K, to the next, L... more
The time course of thermal reactions after illumination of 100% humidified bacteriorhodopsin films was followed with FTIR spectroscopy between 125 and 195 K. We monitored the conversion of the initial photoproduct, K, to the next, L intermediate, and a shunt reaction of the L state directly back to the initial BR state. Both reactions can be described by either multiexponential kinetics, which would lead to apparent end-state mixtures that contain increasing amounts of the product, i.e., L or BR, with increasing temperature, or distributed kinetics. Conventional kinetic schemes that could account for the partial conversion require reversible reactions, branching, or parallel cycles. These possibilities were tested by producing K or L and monitoring their interconversion at a single temperature and by shifting the temperature upward or downward after an initial incubation and after their redistribution. The results are inconsistent with any conventional scheme. Instead, we attribute ...
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Research Interests: Biophysics, Chemistry, Calcium, Biophysical Chemistry, Biological Sciences, and 13 moreMutation, Physical sciences, Bacteriorhodopsin, D-Aspartic Acid, CHEMICAL SCIENCES, Surface Properties, Cysteine, Calorimetry, Halobacterium Salinarum, Protein Binding, Hydrogen-Ion Concentration, Protons, and binding sites
Research Interests: Chemistry, Kinetics, Photochemistry, Biophysical Chemistry, Medicine, and 15 moreBiological Sciences, Physical sciences, Light, Temperature, Temperature Dependence, CHEMICAL SCIENCES, General Electric, Time Dependent, Spectrum analysis, Model Analysis, Protein Conformation, Energy Transfer, Protons, Rhodopsin, and Isomerism
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In the bacteriorhodopsin photocycle the recovery of the initial BR state from the M intermediate occurs via the N and O intermediates. The molecular events in this process include reprotonation of the Schiff base and the subsequent uptake... more
In the bacteriorhodopsin photocycle the recovery of the initial BR state from the M intermediate occurs via the N and O intermediates. The molecular events in this process include reprotonation of the Schiff base and the subsequent uptake of a proton from the cytoplasmic side, as well as reisomerization of the retinal from 13-cis to all-trans. We have studied the kinetics of the intermediates and the proton uptake. At moderately low pH little of the N state accumulates, and the O state dominates in the reactions that lead from M to BR. The proton uptake lags behind the formation of O, suggesting the sequence N(0)<==>O(0) + H+ (from the bulk)-->O(+1)-->BR+H+ (to the bulk), where the superscripts indicate the net protonation state of the protein relative to BR. Together with a parallel study of ours at moderately high pH, these results suggest that the sequence of proton uptake and retinal reisomerization depends on pH: at low pH the isomerization occurs first and O accumulates, but at high pH the isomerization is delayed and therefore N accumulates. Although this model contains too many rate constants for rigorous testing, we find that it will generate most of the characteristic pH-dependent kinetic features of the photocycle with few assumptions other than pH dependency for protonation at the proton release and uptake steps.
Research Interests: Biochemistry, Chemistry, Kinetics, Medicine, Light, and 11 moreTime Factors, Halobacterium Salinarum, Amino Acid Sequence, American Chemical Society, Recombinant Proteins, Hydrogen-Ion Concentration, Cytoplasm, Protons, Biochemistry and cell biology, Isomerism, and Medical biochemistry and metabolomics
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion... more
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding ...
Research Interests: Biochemistry, Biophysics, Chemistry, Kinetics, D-Aspartic Acid, and 13 moreSchiff bases, Sodium, Fourier transform infrared spectroscopy, Amino Acid Sequence, Amino Acid Substitution Rates, Recombinant Proteins, Hydrogen-Ion Concentration, Site-directed Mutagenesis, Biochemistry and cell biology, Photochemical Processes, Molecular Sequence Data, binding sites, and Medical biochemistry and metabolomics
The time course of thermal reactions after illumination of 100% humidified bacteriorhodopsin films was followed with FTIR spectroscopy between 125 and 195 K. We monitored the conversion of the initial photoproduct, K, to the next, L... more
The time course of thermal reactions after illumination of 100% humidified bacteriorhodopsin films was followed with FTIR spectroscopy between 125 and 195 K. We monitored the conversion of the initial photoproduct, K, to the next, L intermediate, and a shunt reaction of the L state directly back to the initial BR state. Both reactions can be described by either multiexponential kinetics, which would lead to apparent end-state mixtures that contain increasing amounts of the product, i.e., L or BR, with increasing temperature, or distributed kinetics. Conventional kinetic schemes that could account for the partial conversion require reversible reactions, branching, or parallel cycles. These possibilities were tested by producing K or L and monitoring their interconversion at a single temperature and by shifting the temperature upward or downward after an initial incubation and after their redistribution. The results are inconsistent with any conventional scheme. Instead, we attribute ...
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Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protonation of Asp-85 during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate)... more
Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protonation of Asp-85 during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pKa of this buried aspartate and those of the other acidic groups. Unlike the case of Glu-204, however, replacement of Glu-194 with aspartate raises the pKa for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm-1. It can be assigned from [4-13C]aspartate and D2O isotope shifts to the C&dbd;O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pKa of about 9. We show that this pKa is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation, while 13C NMR titration indicates that Asp-194 has an initial pKa of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pKa's change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the medium.
Research Interests: Biochemistry, Chemistry, Kinetics, Photochemistry, Medicine, and 14 moreBacteriorhodopsin, Proton Transfer, Schiff bases, Fourier transform infrared spectroscopy, Proton, Protein Conformation, Halobacterium Salinarum, Hydrogen-Ion Concentration, Protons, Glutamic Acid, Biochemistry and cell biology, Cell Membrane, binding sites, and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Chemistry, Kinetics, Medicine, Macromolecular X-Ray Crystallography, and 15 moreLight, Bacteriorhodopsin, D-Aspartic Acid, Protein Secondary Structure Prediction, Fourier transform infrared spectroscopy, Molecular Conformation, Halobacterium Salinarum, Amino Acid Sequence, Amino Acid Substitution Rates, Recombinant Proteins, Hydrogen-Ion Concentration, Biochemistry and cell biology, binding sites, Medical biochemistry and metabolomics, and stereoisomerism
The accessibility of the retinal Schiff base in bacteriorhodopsin was studied in the D85N/D96N mutant where the proton acceptor and donor are absent. Protonation and deprotonation of the Schiff base after pH jump without illumination and... more
The accessibility of the retinal Schiff base in bacteriorhodopsin was studied in the D85N/D96N mutant where the proton acceptor and donor are absent. Protonation and deprotonation of the Schiff base after pH jump without illumination and in the photocycle of the unprotonated Schiff base were measured in the visible and the infrared. Whether access is extracellular (EC) or cytoplasmic (CP) was decided from the effect of millimolar concentrations of azide on the rates of proton transfers. The results, together with earlier work on the wild-type protein, suggest a new hypothesis for the proton-transfer switch: (i) In the metastable 13-cis, 15-anti and all-trans, 15-syn photoproducts, but not in the stable isomeric states, access flickers between the EC and CP directions. (ii) The direction of proton transfer is decided both by this local access and by the presence of a suitable donor or acceptor group (in the wild type), or the proton conductivity in the EC and CP half-channels (in D85N/D96N). (iii) Thermal reisomerization of the retinal can occur only when the Schiff base is protonated, as is well-known. In the wild-type transport cycle, the concurrent local EC and CP access during the lifetime of the metastable 13-cis, 15-anti state enables the changing pKa's of the proton acceptor and donor to determine the direction of proton transfer. Proton transfer from the Schiff base to Asp-85 in the EC direction is followed by reprotonation by Asp-96 from the CP direction because proton release to the EC surface raises the pKa of Asp-85 and a large-scale protein conformation change lowers the pKa of Asp-96. The unexpected finding we report here for D85N/D96N, that when the retinal is in the stable all-trans, 15-anti and 13-cis, 15-syn isomeric forms access of the Schiff base is locked (in the EC and CP directions, respectively), suggests that in this protein reisomerization, rather than changes in the proton conductivities of the EC and CP half-channels, provides the switch function. With this mechanism, the various modes of transport reported for Asp-85 mutants (CP to EC direction with blue light, and EC to CP direction with blue plus green light) are understood also in terms of rules i-iii.
Research Interests: Biochemistry, Chemistry, Kinetics, Photochemistry, Medicine, and 13 moreProton Transfer, D-Aspartic Acid, Electron Transport, Schiff bases, Halobacterium Salinarum, Amino Acid Substitution Rates, Hydrogen-Ion Concentration, Asparagine, Protons, Site-directed Mutagenesis, Biochemistry and cell biology, Isomerism, and Medical biochemistry and metabolomics
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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... more
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°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.
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Proton transfer is crucial for many enzyme reactions. Here, we show that in addition to protonatable amino acid side chains, water networks could constitute proton-binding sites in proteins. A broad IR continuum absorbance change during... more
Proton transfer is crucial for many enzyme reactions. Here, we show that in addition to protonatable amino acid side chains, water networks could constitute proton-binding sites in proteins. A broad IR continuum absorbance change during the proton pumping photocycle of bacteriorhodopsin (bR) indicates most likely deprotonation of a protonated water cluster at the proton release site close to the surface. We investigate the influence of several mutations on the proton release network and the continuum change, to gain information about the location and extent of the protonated water network and to reveal the participating residues necessary for its stabilization. We identify a protonated water cluster consisting in total of one proton and about five water molecules surrounded by six side chains and three backbone groups (Tyr-57, Arg-82, Tyr-83, Glu-204, Glu-194, Ser-193, Pro-77, Tyr-79, and Thr-205). The observed perturbation of proton release by many single-residue mutations is now e...
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Research Interests: Biochemistry, Chemistry, Carotenoids, Medicine, Molecular Mechanics, and 15 moreCrystal structure, Nitrogen, Oscillations, Spectrophotometry, Schiff bases, Hydrogen Bond, Schiff Base, Energy Transfer, Amino Acid Substitution Rates, Protons, Binding Site, Rhodopsin, Biochemistry and cell biology, Configuration Interaction, and Medical biochemistry and metabolomics
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The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were... more
The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.
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Research Interests: Biochemistry, Chemistry, Kinetics, D-Aspartic Acid, Glutamine, and 10 moreFourier transform infrared spectroscopy, Time Factors, Photoperiod, Photolysis, Halobacterium Salinarum, Protons, Glutamic Acid, Biochemistry and cell biology, Fourier transform infrared, and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Chemistry, Water, Kinetics, Medicine, and 14 moreProton Transfer, D-Aspartic Acid, Kinetic Isotope Effect, Arginine, Photolysis, Halobacterium Salinarum, Protons, Site-directed Mutagenesis, Glutamic Acid, Tyrosine, Biochemistry and cell biology, Purple Membrane, Medical biochemistry and metabolomics, and Deuterium oxide
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In the last few years, detailed structural information from high-resolution x-ray diffraction has been added to the already large body of spectroscopic and mutational data on the bacteriorhodopsin proton transport cycle. Although there... more
In the last few years, detailed structural information from high-resolution x-ray diffraction has been added to the already large body of spectroscopic and mutational data on the bacteriorhodopsin proton transport cycle. Although there are still many gaps, it is now possible to reconstruct the main events in the translocation of the proton and how they are coupled to the photoisomerization of the retinal chromophore. Future structural work will concentrate on describing the details of the individual proton transfer steps during the photocycle.
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A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform lightdriven sodium ion transport,... more
A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform lightdriven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na. The absorption spectrum of GLR is insensitive to Na at concentrations of ≤3 M. However, very low concentrations of Na cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a “Na-independent” to a “Na-dependent” photocycle (or photocycle branch) at ∼60 μM Na. The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na concentration. This suggests that a high-affinity Na binding site is created transiently after photoexcita...
ABSTRACT A 2005 januárjában kezdődőtt négy éves OTKA támogatással a membrán fehérjék szerkezete és működése közti kapcsolatok tisztázására folytatott kutatás két fő irányba haladt. Az egyik a membrán fehérjék és itt főleg a fényhajtotta... more
ABSTRACT A 2005 januárjában kezdődőtt négy éves OTKA támogatással a membrán fehérjék szerkezete és működése közti kapcsolatok tisztázására folytatott kutatás két fő irányba haladt. Az egyik a membrán fehérjék és itt főleg a fényhajtotta retinál fehérjék működésével kapcsolatos kutatásokra összpontosított. Ennek során nemcsak a jól ismert retinál fehérjék a bakteriorodopszin, vagy halorodopszin, hanem újonnan felfedezett fehérjék, mint például a xantorodopsin és leptospheria rodopszin működését is tanulmányoztuk, abszorpciókinetikai mérésekkel. A retinál fehérjék kutatásával kapcsolatosan, az OTKA pályázat támogatásával, nyolc cikket közöltünk. Hasonló kinetikai méréseket végeztünk fotoszintetikus rekciócentrumok és szén nanocsövek keverékén, ami ígéretes biotechnologiai anyagnak bizonyúl. A reakciócentrumokkal kapcsolatban két OTKA támogatta cikkünk jelent meg A másik fő kutatási irány a fehérjék, membránok és élő sejtek morfológiai és mechanikai vizsgálata atomerő mikroszkóppal. Természetes és mesterséges membránokon is végeztünk méréseket. Amíg különböző letapogatási technikákkal nagyfelbontású képet alkottunk a tanulmányozott objektumok felszínéről, addig erőméréssel sikerült meghatározni a tanulmányozott minta Young moduluszát és ennek a változását különböző külső körülmények között. Egy teljesen új technikát dolgoztunk ki retinál fehérjék konformációváltozásának atomerőmikroszkópos megfigyelésére. Az elmúlt négy évben ezekkel a mérésekkel kapcsolatban nyolc OTKA által támogatott cikkelt közöltünk. | Beginning in 2005 the four years OTKA supported study of the relation between the structure and function of membrane proteins was conducted in two main directions. One research direction concentrates on the function of the membrane proteins and mainly on the function of the light activated retinal proteins. In this study not only the well known bacteriorhodopsin and halorhodopsin, but some newly discovered proteins, such as the xantorhodopsin and leptospheria rhodopsin were studied by absorption kinetic measurements. Related to this research, eight papers supported by the OTKA were published. Similar kinetic measurements were performed on the mixture of photosynthetic reaction centers and carbon nanotubes. This material is considered as a promising biotechnological material. The results of the reaction center research were published in two OTKA supported papers. The second main research direction was the morphological and mechanical study of the proteins, membranes and living cells by atomic force microscopy. We performed measurements on both artificial and natural membranes. With different scanning techniques high resolution images of the studied object surface were recorded. With force measurement it was possible to determine the Young?s module of the sample and its dependence from the external conditions. A new technique was developed to study the conformational change of the retinal proteins with the atomic force microscope. During the OTKA supported four years, related to this field, eight papers were published.
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A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is... more
A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fra...
Research Interests: Chemistry, Kinetics, Biological Chemistry, Medicine, Biological Sciences, and 15 moreEscherichia coli, Light, Liposomes, Absorption, Schiff bases, CHEMICAL SCIENCES, Lysine, Time Factors, Alanine, Amino Acid Substitution Rates, Hydrogen-Ion Concentration, Protons, Halobacterium, Medical and Health Sciences, and Deuterium oxide
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The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were... more
The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.
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ABSTRACT
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ABSTRACT
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The photocycles of wild-type bacteriorhodopsin and its D96N form were investigated with a gated multichannel analyzer. Reconstruction of the spectra of the photointermediates from the measured time-resolved difference spectra allowed... more
The photocycles of wild-type bacteriorhodopsin and its D96N form were investigated with a gated multichannel analyzer. Reconstruction of the spectra of the photointermediates from the measured time-resolved difference spectra allowed evaluation of the kinetics; the data at pH 7 in the presence of 100 mM NaCl were best fitted by the scheme K in eqiulibrium L in equilibrium M1----M2 in equilibrium N in equilibrium O----BR plus N----BR [Váró, G., & Lanyi, J. K. (1990) Biochemistry 29, 2241-2250]. The proposed two M states and the M1----M2 reaction were necessitated by anomalies in the kinetics of the decay of K and L. Additional support was provided by a 4-nm blue-shift in the maximum of M in Triton X-100 solubilized bacteriorhodopsin during the photocycle; the kinetics of the shift were consistent with the time course of the proposed M1----M2 transition. In the D96N mutant, the M state is stabilized, and the resulting equilibrium mixture for the intermediates could be evaluated with greater precision. The concentration ratio of L to M at the equilibrium was estimated to be no higher than 0.01. This requires the ratio of forward/reverse rates for the M1 to M2 conversion to be at least 200, i.e., a virtually irreversible reaction. Consistent with an earlier report, the data at lower pH and in the absence of NaCl are different and suggest the existence of a second L species; we propose that it is in equilibrium with M2.