المؤلفون: David Jacobson, Thomas T. Perkins

المصدر: Proc Natl Acad Sci U S A

مصطلحات موضوعية: Halobacterium salinarum, Protein Folding, Multidisciplinary, biology, Chemistry, Bilayer, Lipid Bilayers, Force spectroscopy, Energy landscape, Bacteriorhodopsin, Biological Sciences, Microscopy, Atomic Force, Single Molecule Imaging, Amino Acid Substitution, Membrane protein, Bacteriorhodopsins, biology.protein, Biophysics, Point Mutation, Thermodynamics, Protein folding, Denaturation (biochemistry), Protein secondary structure

الوصف: Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔG for mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val(217) and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔG for a fully folded membrane protein embedded in its native bilayer.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e69915c7478f1a6efdaf795b251d3b01Test
https://doi.org/10.1073/pnas.2020083118Test

المؤلفون: Marilyn R. Gunner, Yifan Song

المصدر: Proceedings of the National Academy of Sciences. 111:16377-16382

مصطلحات موضوعية: Halobacterium salinarum, Models, Molecular, Protein Conformation, Molecular Sequence Data, Static Electricity, Protonation, Reaction intermediate, Molecular Dynamics Simulation, Residue (chemistry), Ion binding, Chlorides, Species Specificity, Catalytic Domain, Amino Acid Sequence, Amino Acids, Conserved Sequence, Binding Sites, Ion Transport, Multidisciplinary, Sequence Homology, Amino Acid, biology, Chemistry, Bacteriorhodopsin, Biological Sciences, Halorhodopsin, Crystallography, Amino Acid Substitution, Ion pump, Bacteriorhodopsins, biology.protein, Proton affinity, Protons, Halorhodopsins, Monte Carlo Method, Sequence Alignment

الوصف: Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl(-) is bound to HR in a position near the deleted acid. In the HR ground state, Cl(-) binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl(-) from the central T111 site and the tight binding of Cl(-) to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::51ac48db7072c2f782b2b3a0ae3d5bffTest
https://doi.org/10.1073/pnas.1411119111Test

المؤلفون: Erik Freier, Steffen Wolf, Klaus Gerwert

المصدر: Proceedings of the National Academy of Sciences. 108:11435-11439

مصطلحات موضوعية: Halobacterium salinarum, Models, Molecular, Conformational change, Protein Conformation, Protonation, Molecular Dynamics Simulation, Biophysical Phenomena, Molecular dynamics, Protein structure, Spectroscopy, Fourier Transform Infrared, Water cluster, Binding Sites, Multidisciplinary, biology, Chemistry, Membrane Proteins, Water, Hydrogen Bonding, Bacteriorhodopsin, Biological Sciences, Proton pump, Crystallography, Membrane, Amino Acid Substitution, Bacteriorhodopsins, Mutagenesis, Site-Directed, biology.protein, Protons, Hydrophobic and Hydrophilic Interactions, Protein Binding

الوصف: High-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water molecules. Their possible active involvement in protein function has recently come into focus. An illustration of the formation of a protonated protein-bound water cluster that is actively involved in proton transfer was described for the membrane protein bacteriorhodopsin (bR) [Garczarek F, Gerwert K (2006) Nature 439:109–112]. Here we show through a combination of time-resolved FTIR spectroscopy and molecular dynamics simulations that three protein-bound water molecules are rearranged by a protein conformational change that resulted in a transient Grotthuss-type proton-transfer chain extending through a hydrophobic protein region of bR. This transient linear water chain facilitates proton transfer at an intermediate conformation only, thereby directing proton transfer within the protein. The rearrangement of protein-bound water molecules that we describe, from inactive positions in the ground state to an active chain in an intermediate state, appears to be energetically favored relative to transient incorporation of water molecules from the bulk. Our discovery provides insight into proton-transfer mechanisms through hydrophobic core regions of ubiquitous membrane spanning proteins such as G-protein coupled receptors or cytochrome C oxidases.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::dfbde4fc7d4da47f50011cc8ab3c9540Test
https://doi.org/10.1073/pnas.1104735108Test

المؤلفون: Prasad Phatak, Marcus Elstner, Nilanjan Ghosh, Haibo Yu, Qiang Cui

المصدر: Proceedings of the National Academy of Sciences. 105:19672-19677

مصطلحات موضوعية: Models, Molecular, Spectrophotometry, Infrared, Proton, Static Electricity, Glutamic Acid, Infrared spectroscopy, Crystallography, X-Ray, Protein Structure, Secondary, Delocalized electron, Computer Simulation, Water cluster, Spectroscopy, Multidisciplinary, biology, Hydrogen bond, Chemistry, Intermolecular force, Hydrogen Bonding, Bacteriorhodopsin, Crystallography, Bacteriorhodopsins, Mutation, Physical Sciences, biology.protein, Quantum Theory, Protons

الوصف: The positions of protons are not available in most high-resolution structural data of biomolecules, thus the identity of proton storage sites in biomolecules that transport proton is generally difficult to determine unambiguously. Using combined quantum mechanical/molecular mechanical computations, we demonstrate that a pair of conserved glutamate residues (Glu 194/204) bonded by a delocalized proton is the proton release group that has been long sought in the proton pump, bacteriorhodopsin. This model is consistent with all available experimental structural and infrared data for both the wild-type bacteriorhodopsin and several mutants. In particular, the continuum infrared band in the 1,800- to 2,000-cm −1 region is shown to arise due to the partially delocalized nature of the proton between the glutamates in the wild-type bacteriorhodopsin; alternations in the flexibility of the glutamates and electrostatic nature of nearby residues in various mutants modulate the degree of proton delocalization and therefore intensity of the continuum band. The strong hydrogen bond between Glu 194/204 also significantly shifts the carboxylate stretches of these residues well −1 , which explains why carboxylate spectral shift was not observed experimentally in the typical >1,700-cm −1 region upon proton release. By contrast, simulations with the proton restrained on the nearby water cluster, as proposed by several recent studies [see, for example, Garezarek K, Gerwert K (2006) Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy. Nature 439:109], led to significant structural deviations from available X-ray structures. This study establishes a biological function for strong, low-barrier hydrogen bonds.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c9f83f1d8473bf702f148a93294ef1b2Test
https://doi.org/10.1073/pnas.0810712105Test

المؤلفون: Vikram S. Bajaj, Judith Herzfeld, M.K. Hornstein, Robert G. Griffin, Melody L. Mak-Jurkauskas, Marina Belenky

المصدر: Proceedings of the National Academy of Sciences. 105:883-888

مصطلحات موضوعية: Halobacterium salinarum, chemistry.chemical_classification, Magnetic Resonance Spectroscopy, Time Factors, Multidisciplinary, Schiff base, Molecular Structure, Double bond, biology, Photochemistry, Bacteriorhodopsin, Nuclear magnetic resonance spectroscopy, Crystallography, chemistry.chemical_compound, Solid-state nuclear magnetic resonance, chemistry, Bacteriorhodopsins, Physical Sciences, biology.protein, Molecule, Single bond, Counterion

الوصف: By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The 15 N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult, as judged by the multiple L substates, with weaker counterion interactions, that are trapped at lower temperatures. The double-bond torsion ultimately developed in the first half of the photocycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::39b81601ab9e44c7a1a60687b0548dfdTest
https://doi.org/10.1073/pnas.0706156105Test

المؤلفون: Andrei K. Dioumaev, Janos K. Lanyi

المصدر: Proceedings of the National Academy of Sciences. 104:9621-9626

مصطلحات موضوعية: Multidisciplinary, Light, biology, Photochemistry, Protein Conformation, Chemistry, Kinetics, Biophysics, Temperature, Analytical chemistry, Bacteriorhodopsin, Biological Sciences, Branching (polymer chemistry), Kinetic energy, Biophysical Phenomena, Reversible reaction, chemistry.chemical_compound, Protein structure, Myoglobin, Chemical physics, Bacteriorhodopsins, Spectroscopy, Fourier Transform Infrared, biology.protein, Redistribution (chemistry)

الوصف: 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 the partial conversions to the other alternative, distributed kinetics, observed previously in myoglobin, which arise from an ensemble of frozen conformational substates at the cryogenic temperatures. In this case, the time course of the reactions reflects the progressive depletion of distinct microscopic substates in the order of their increasing activation barriers, with a distribution width for K to L reaction of ≈7 kJ/mol.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4b6ba1c8f7f94c039a17425e60178010Test
https://doi.org/10.1073/pnas.0703859104Test

المؤلفون: Yuki Sudo, John L. Spudich

المصدر: Proceedings of the National Academy of Sciences. 103:16129-16134

مصطلحات موضوعية: Halobacterium salinarum, Models, Molecular, Sensory Receptor Cells, Photoisomerization, Photochemistry, Crystallography, X-Ray, Sensory Rhodopsins, Protein structure, Phototaxis, Multidisciplinary, biology, Chemistry, Spectrum Analysis, Sensory rhodopsin II, Hydrogen Bonding, Bacteriorhodopsin, Biological Sciences, biology.organism_classification, Protein Structure, Tertiary, Protein Transport, Crystallography, Rhodopsin, Bacteriorhodopsins, biology.protein, Biophysics, Protons, Protein Binding

الوصف: In haloarchaea, light-driven ion transporters have been modified by evolution to produce sensory receptors that relay light signals to transducer proteins controlling motility behavior. The proton pump bacteriorhodopsin and the phototaxis receptor sensory rhodopsin II (SRII) differ by 74% of their residues, with nearly all conserved residues within the photoreactive retinal-binding pocket in the membrane-embedded center of the proteins. Here, we show that three residues in bacteriorhodopsin replaced by the corresponding residues in SRII enable bacteriorhodopsin to efficiently relay the retinal photoisomerization signal to the SRII integral membrane transducer (HtrII) and induce robust phototaxis responses. A single replacement (Ala-215–Thr), bridging the retinal and the membrane-embedded surface, confers weak phototaxis signaling activity, and the additional two (surface substitutions Pro-200–Thr and Val-210–Tyr), expected to align bacteriorhodopsin and HtrII in similar juxtaposition as SRII and HtrII, greatly enhance the signaling. In SRII, the three residues form a chain of hydrogen bonds from the retinal's photoisomerized C 13 C 14 double bond to residues in the membrane-embedded α-helices of HtrII. The results suggest a chemical mechanism for signaling that entails initial storage of energy of photoisomerization in SRII's hydrogen bond between Tyr-174, which is in contact with the retinal, and Thr-204, which borders residues on the SRII surface in contact with HtrII, followed by transfer of this chemical energy to drive structural transitions in the transducer helices. The results demonstrate that evolution accomplished an elegant but simple conversion: The essential differences between transport and signaling proteins in the rhodopsin family are far less than previously imagined.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::39eae9544fdf8413eebf6bbf70609d09Test
https://doi.org/10.1073/pnas.0607467103Test

المؤلفون: Bernd Simon, Ronald Kühne, Peter Schmieder, Dieter Oesterhelt, Brigitte Kessler, Heiko Patzelt, Antonius terLaak, Hartmut Oschkinat

المصدر: Proceedings of the National Academy of Sciences of the United States of America, 99(15): 9765-9770

مصطلحات موضوعية: Models, Molecular, Binding Sites, Magnetic Resonance Spectroscopy, Multidisciplinary, Schiff base, biology, Protein Conformation, Chemistry, Bacteriorhodopsin, Nuclear magnetic resonance spectroscopy, Biological Sciences, Darkness, Chromophore, Dipole, chemistry.chemical_compound, Crystallography, Deprotonation, Protein structure, Catalytic cycle, Bacteriorhodopsins, biology.protein, Amino Acid Sequence

الوصف: The two forms of bacteriorhodopsin present in the dark-adapted state, containing either all-trans or 13- cis ,15- syn retinal, were examined by using solution state NMR, and their structures were determined. Comparison of the all-trans and the 13- cis ,15- syn forms shows a shift in position of about 0.25 Å within the pocket of the protein. Comparing this to the 13- cis ,15- anti chromophore of the catalytic cycle M-intermediate structure, the 13- cis ,15- syn form demonstrates a less pronounced up-tilt of the retinal C12—C14 region, while leaving W182 and T178 essentially unchanged. The N—H dipole of the Schiff base orients toward the extracellular side in both forms, however, it reorients toward the intracellular side in the 13- cis ,15- anti configuration to form the catalytic M-intermediate. Thus, the change of the N—H dipole is considered primarily responsible for energy storage, conformation changes of the protein, and the deprotonation of the Schiff base. The structural similarity of the all-trans and 13- cis ,15- syn forms is taken as strong evidence for the ion dipole dragging model by which proton (hydroxide ion) translocation follows the change of the dipole.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::1afa9f834aa8133fd15318f0d87b0569Test
https://doi.org/10.1073/pnas.132253899Test

المؤلفون: Stephan L. Logunov, Markus Braun, Mostafa A. El-Sayed, Victor Volkov

المصدر: Proceedings of the National Academy of Sciences. 98:8475-8479

مصطلحات موضوعية: Halobacterium salinarum, education.field_of_study, Multidisciplinary, Chemistry, Population, Relaxation (NMR), Conical intersection, Photochemistry, Laser, law.invention, Spectrophotometry, law, Bacteriorhodopsins, Excited state, Physical Sciences, Femtosecond, Retinaldehyde, Atomic physics, education, Ground state, Excitation

الوصف: We present the results of two-pump and probe femtosecond experiments designed to follow the relaxation dynamics of the lowest excited state (S 1 ) populated by different modes. In the first mode, a direct (S 0 → S 1 ) radiative excitation of the ground state is used. In the second mode, an indirect excitation is used where the S 1 state is populated by the use of two femtosecond laser pulses with different colors and delay times between them. The first pulse excites the S 0 → S 1 transition whereas the second pulse excites the S 1 → S n transition. The nonradiative relaxation from the S n state populates the lowest excited state. Our results suggest that the S 1 state relaxes faster when populated nonradiatively from the S n state than when pumped directly by the S 0 → S 1 excitation. Additionally, the S n → S 1 nonradiative relaxation time is found to change by varying the delay time between the two pump pulses. The observed dependence of the lowest excited state population as well as its dependence on the delay between the two pump pulses are found to fit a kinetic model in which the S n state populates a different surface (called S′ 1 ) than the one being directly excited (S 1 ). The possible involvement of the A g type states, the J intermediate, and the conical intersection leading to the S 0 or to the isomerization product (K intermediate) are discussed in the framework of the proposed model.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7c509d3796b8f25e034a168f20c76909Test
https://doi.org/10.1073/pnas.141220198Test

المؤلفون: Judith Herzfeld, Jan Raap, Johan Lugtenburg, Hideki Kandori, Yoichi Yamazaki, Yoshinori Shichida, Marina Belenky

المصدر: Proceedings of the National Academy of Sciences. 98:1571-1576

مصطلحات موضوعية: Threonine, Aspartic Acid, Multidisciplinary, Schiff base, biology, Photoisomerization, Hydrogen bond, Membrane Proteins, Active site, Bacteriorhodopsin, Oxygen Isotopes, Proton Pumps, Biological Sciences, Chromophore, Acceptor, chemistry.chemical_compound, Crystallography, chemistry, Bacteriorhodopsins, Isotope Labeling, Proton transport, biology.protein

الوصف: Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O—H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3- 18 O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O—D group of Thr-89 in D 2 O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O—H (O—D) group of Thr-89 in M is indicated by a shift in the C⩵O stretching vibration of the former because of 18 O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ebad6b41d5755db8eeeb4129c79965c0Test
https://doi.org/10.1073/pnas.98.4.1571Test