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Janos K Lanyi

Publications and source records attributed to Janos K Lanyi.

17 recordsLinked to original sources
PubMed2005

Proton binding within a membrane protein by a protonated water cluster.

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 explained by the influence of numerous side chains on the protonated H bonded network. In situ hydrogen/deuterium exchange Fourier transform IR measurements of the bR ground state, show that the proton of the release group becomes localized on Glu-204 and Asp-204 in the ground state of the mutants E194D and E204D, respectively, even though it is delocalized in the ground state of wild-type bR. Thus, the release mechanism switches between the wild-type and mutated proteins from a delocalized to a localized proton-binding site.

Binding Sites↗
PubMed2004

What is the real crystallographic structure of the L photointermediate of bacteriorhodopsin?

In the last few years, three laboratories have reported three entirely different crystallographic models for the L photointermediate of bacteriorhodopsin. All are from X-ray diffraction of illuminated crystals that contain L in photostationary states created at similar cryogenic temperatures. This article compares the models and their implications, the crystallographic statistics and the methods used to derive them, as well as their agreement with non-crystallographic information.

Bacteriorhodopsins↗
PubMed2004

Selectivity of retinal photoisomerization in proteorhodopsin is controlled by aspartic acid 227.

Similarly to bacteriorhodopsin, proteorhodopsin that normally contains all-trans and 13-cis retinal is transformed at low pH to a species containing 9-cis retinal under continuous illumination at lambda > 530 nm. This species, absorbing around 430 nm, returns thermally in tens of minutes to initial pigment and can be reconverted also with blue-light illumination. The yield of the 9-cis species is negligibly small at neutral pH but increases manyfold (>100) at acid pH with a pK(a) of 2.6. This indicates that protonation of acidic group(s) alters the photoreaction pathway that leads normally to all-trans --> 13-cis isomerization. In the D97N mutant, in which one of the two acidic groups in the vicinity of the retinal Schiff base is not ionizable, the yield of 9-cis species at low pH shows a pH dependence similar to that in the wild-type but with a somewhat increased pK(a) of 3.3. In contrast to this relatively minor effect, replacement of the other acidic group, Asp227, with Asn results in a remarkable, more than 50-fold, increase in the yield of the light-induced formation of 9-cis species in the pH range 4-6. It appears that protonation of Asp227 at low pH is what causes the dramatic increase in the yield of the 9-cis species in wild-type proteorhodopsin. We conclude that the photoisomerization pathways in proteorhodopsin to 13-cis or 9-cis photoproducts are controlled by the charge state of Asp227.

Amino Acid Substitution↗
PubMed2004

Local-global conformational coupling in a heptahelical membrane protein: transport mechanism from crystal structures of the nine states in the bacteriorhodopsin photocycle.

Proton pumps utilize a chemical or photochemical reaction to create pH and electrical gradients between the interior and the exterior of cells and organelles that energize ATP synthesis and the accumulation and extrusion of solutes and ions. G-protein coupled receptors bind agonists and assume signaling states that communicate with the coupled transducers. How these two kinds of proteins convert chemical potential to a proton transmembrane electrochemical potential or a signal are the great questions in structural membrane biology, and they may have a common answer. Bacteriorhodopsin, a particularly simple integral membrane protein, functions as a proton pump but has a heptahelical structure like membrane receptors. Crystallographic structures are now available for all of the intermediates of the bacteriorhodopsin transport cycle, and they describe the proton translocation mechanism, step by step and in atomic detail. The results show how local conformational changes propagate upon the gradual relaxation of the initially twisted photoisomerized retinal toward the two membrane surfaces. Such local-global conformational coupling between the ligand-binding site and the distant regions of the protein may be the shared mechanism of ion pumps and G-protein related receptors.

Bacteriorhodopsins↗
PubMed2004

The nitrate transporting photochemical reaction cycle of the pharaonis halorhodopsin.

Time-resolved spectroscopy, absorption kinetic and electric signal measurement techniques were used to study the nitrate transporting photocycle of the pharaonis halorhodopsin. The spectral titration reveals two nitrate-binding constants, assigned to two independent binding sites. The high-affinity binding site (K(a) = 11 mM) contributes to the appearance of the nitrate transporting photocycle, whereas the low-affinity constant (having a K(a) of approximately 7 M) slows the last decay process in the photocycle. Although the spectra of the intermediates are not the same as those found in the chloride transporting photocycle, the sequence of the intermediates and the energy diagrams are similar. The differences in spectra and energy levels can be attributed to the difference in the size of the transported chloride or nitrate. Electric signal measurements show that a charge is transferred across the membrane during the photocycle, as expected. A new observation is an apparent release and rebinding of a small fraction of the retinal, inside the retinal pocket, during the photocycle. The release occurs during the N-to-O transition, whereas the rebinding happens in several seconds, well after the other steps of the photocycle are over.

Biological Transport, Active↗
PubMed2004

Bacteriorhodopsin.

Fourier transform infrared and Raman spectroscopy, solid-state NMR, and X-ray crystallography have contributed detailed information about the structural changes in the proton transport cycle of the light-driven pump, bacteriorhodopsin. The results over the past few years add up to a step-by-step description of the configurational changes of the photoisomerized retinal, how these changes result in internal proton transfers and the release of a proton to the extracellular surface and uptake on the other side, as well as the conservation and transformation of excess free energy during the cycle.

Bacteriorhodopsins↗
PubMed2003

Crystallographic structures of the M and N intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water molecules between Asp-96 and the retinal Schiff base.

An M intermediate of wild-type bacteriorhodopsin and an N intermediate of the V49A mutant were accumulated in photostationary states at pH 5.6 and 295 K, and their crystal structures determined to 1.52A and 1.62A resolution, respectively. They appear to be M(1) and N' in the sequence, M(1)<-->M(2)<-->M'(2)<-->N<-->N'-->O-->BR, where M(1), M(2), and M'(2) contain an unprotonated retinal Schiff base before and after a reorientation switch and after proton release to the extracellular surface, while N and N' contain a reprotonated Schiff base, before and after reprotonation of Asp96 from the cytoplasmic surface. In M(1), we detect a cluster of three hydrogen-bonded water molecules at Asp96, not present in the BR state. In M(2), whose structure we reported earlier, one of these water molecules intercalates between Asp96 and Thr46. In N', the cluster is transformed into a single-file hydrogen-bonded chain of four water molecules that connects Asp96 to the Schiff base. We find a network of three water molecules near residue 219 in the crystal structure of the non-illuminated F219L mutant, where the residue replacement creates a cavity. This suggests that the hydration of the cytoplasmic region we observe in N' might have occurred spontaneously, beginning at an existing water molecule as nucleus, in the cavities from residue rearrangements in the photocycle.

Aspartic Acid↗
PubMed2003

Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic.

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.

Aspartic Acid↗
PubMed2003

Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K, L, M1, M2, and M2' intermediates of the photocycle.

We produced the L intermediate of the photocycle in a bacteriorhodopsin crystal in photo-stationary state at 170 K with red laser illumination at 60% occupancy, and determined its structure to 1.62 A resolution. With this model, high-resolution structural information is available for the initial bacteriorhodopsin, as well as the first five states in the transport cycle. These states involve photo-isomerization of the retinal and its initial configurational changes, deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. The six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base.

Bacteriorhodopsins↗
PubMed2003

The photochemical reaction cycle of proteorhodopsin at low pH.

The proton acceptor group in the recently described retinal protein, proteorhodopsin has an unusually high pK(a) of 7.1. It was shown that at pH above this pK(a), illumination initiates a photocycle similar to that of bacteriorhodopsin, and the protein transports proton across the cell membrane. Recently it was reported that proteorhodopsin, unlike bacteriorhodopsin, transports protons at pH below the pK(a) of the proton acceptor, and this transport is in the reverse direction. We have investigated the photocycle of proteorhodopsin at such low pH. At pH 5, three spectrally distinct intermediates K, L, and N, and another spectrally silent one, PR', could be identified, but a deprotonated Schiff base containing M-like intermediate, characteristic for proton pumping activity, does not accumulate. All the reactions between the intermediates are close to equilibrium, except the last transition from PR' to PR, when the protein returns to its initial unexcited state in a quasiunidirectional reaction. The electric signal measurements indicate that although charge motions are detected inside the protein, their net dislocation is zero, indicating that contrary to the earlier reported, at low pH no charged particle is transported across the membrane.

Hydrogen-Ion Concentration↗
PubMed2003

Characterization of the photochemical reaction cycle of proteorhodopsin.

Absorption changes in the photocycle of the recently described retinal protein, proteorhodopsin, are analyzed. The transient spectra at pH 9.5, where it acts as a light-driven proton pump, reveal the existence of three spectrally different intermediates, K, M, and N, named in analogy with the photointermediates of bacteriorhodopsin. Model analysis based on time-dependent absorption kinetic signals at four wavelengths suggested the existence of two more spectrally silent intermediates and lead to a sequential reaction scheme with five intermediates, K, M(1), M(2), N, and PR', before decay to the initial state PR. An L-like intermediate was not observed, probably for kinetic reasons. By measuring the light-generated electric signal of an oriented sample, the electrogenicity of each intermediate could be determined. The electrogenicities of the first three intermediates (K, M(1), and M(2)) have small negative value, but the last three components, corresponding to the N and PR' intermediates and PR, are positive and two-orders-of-magnitude larger. These states give the major contributions to the proton translocation across the membrane. The energetic scheme of the photocycle was calculated from the temperature-dependence of the absorption kinetic signals.

Energy Transfer↗
PubMed2002

Changes in hydrogen bonding and environment of tryptophan residues on helix F of bacteriorhodopsin during the photocycle: a time-resolved ultraviolet resonance Raman study.

Protein structural changes during the photocycle of bacteriorhodopsin were examined by time-resolved ultraviolet resonance Raman (UVRR) spectroscopy. Most of the 244-nm UVRR difference signals of Trp were assigned to either Trp182 or Trp189 using the Trp182 --> Phe and Trp189 --> Phe mutants. The W17 mode of Trp182 shows a wavenumber downshift in the M(1) --> M(2) transition, indicating an increase in hydrogen bonding strength at the indole nitrogen. On the other hand, Trp189 shows Raman intensity increases of the W16 and W18 modes ascribable to an increased hydrophobic interaction. These observations suggest that the tilt of helix F, which ensures that reprotonation of the Schiff base is from the cytoplasmic side, occurs in the M(1) --> M(2) transition. In the M(2) --> N transition, the environment of Trp189 returns to the initial state, whereas the hydrophobic interaction of Trp182 decreases drastically. The decrease in hydrophobic interaction of Trp182 in the N state suggests an invasion of water molecules that promote the proton transfer from Asp96 to the Schiff base. Structural reorganization of the protein after the tilt of helix F may be important for efficient reprotonation of the Schiff base.

Bacteriorhodopsins↗
PubMed2002

Proton transfers in the photochemical reaction cycle of proteorhodopsin.

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.

Bacteriorhodopsins↗
PubMed2002

Conformational change of the E-F interhelical loop in the M photointermediate of bacteriorhodopsin.

The conformation of the structured EF interhelical loop of bacteriorhodopsin and its change in the M photointermediate were assessed by measuring the rate of reaction of 16 single engineered cysteine residues along the loop with water-soluble sulfhydryl reagents. The exposure to the bulk in the unilluminated state determined with the cysteine reaction correlated well with the degree of access to water calculated from the crystallographic structure of the loop. The EF-loop should be affected by the well-known outward tilt of helix F in the M and N intermediates of the photocycle. A second mutation in each cysteine mutant, the D96N residue replacement, allowed full conversion to the M state by illumination. The reaction rates measured under these conditions indicated that buried residues tend to become more exposed, and exposed residues become more buried in M. This is to be expected from tilt of helix F. However, the observation of increased exposure of four residues near the middle of the loop, where steric effects are only from other loop residues, indicate that the conformation of the EF-loop itself is changed. Thus, the motion of the loop in M is more complex than expected from simple tilt of helix F, and may include rotation that unwinds its twist.

Bacteriorhodopsins↗
PubMed2002

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin.

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.

Absorption↗
PubMedDate not supplied

X-ray diffraction of bacteriorhodopsin photocycle intermediates.

Recent advances in the crystallography of bacteriorhodopsin, the light-driven proton pump, have yielded structural models for all intermediates of the photochemical cycle. For seven of the species, X-ray diffraction data were collected from trapped photostationary states in crystals, and for the two remaining ones the structures of selected mutants are available. The changes of the retinal chromophore, protein and bound water describe, at an atomic level, how accommodation of the twisted photoisomerized retinal to its binding site causes de-protonation of the retinal Schiff base and initiates cascades of gradual conformational rearrangements of the protein. One cascade propagates in the extracellular direction and results in proton release, and the other in the cytoplasmic direction and results in side-chain and main-chain rearrangements, formation of a chain of hydrogen-bonded water, and proton uptake from the bulk. Such local-global conformational coupling, with gradual spreading of a local perturbation over the rest of the protein, might be the uniting principle of transporters and receptors.

Bacteriorhodopsins↗