bR Ground State Research Articles

A vector correlation study using a hexapole-oriented molecular beam: photodissociation dynamics of oriented isohaloethane.

The photodissociation dynamics of isohaloethane (1-bromo-2-chloro-1,1,2-trifluoroethane) at 234 nm was studied by a sliced imaging technique combined with an oriented molecular beam. The speed and angular distributions of the competitive products of spin-orbit selected Br and Cl atoms were determined by analysis of the obtained images. The anisotropic parameter, β, was found to be 2.0 ± 0.2 for the excited state of Br(2P1/2) (Br*) and 1.2 ± 0.3 for the ground state of Br(2P3/2) (Br). The speed distributions for both Br and Br* exhibited Gaussian-like characteristics. These results indicate that Br atoms were generated by direct formation after excitation through the nσ*(C-Br) potential energy surfaces. In contrast, the angular distributions for the Cl fragments were almost isotropic, while the speed distributions displayed Boltzmann-like characteristics. This suggests that the Cl atoms may form through long-lived parent molecules after photoexcitation. The branching ratio for Br and Cl atom formation was found to be approximately 1.2, that is, Br atom formation occurred preferentially, in contrast to the case of halothane photodissociation reported in our previous work [Che et al., J. Phys. Chem. A, 2020, 124, 5288]. A vector correlation study between the laser polarization axis and the direction of the dipole moment revealed a similar tendency for all photofragments, suggesting that the fragments were formed through a common excited state of isohaloethane. The vector correlation was also studied theoretically for comparison with the experimental results. The angle between the transition dipole moment in photodissociation and the permanent dipole moment was found to be 42 ± 15°. The obtained results indicate that this vector correlation approach combined with an oriented molecular beam is a powerful tool for determining the transition dipole moments in photodissociation.

O to bR transition in bacteriorhodopsin occurs through a proton hole mechanism

Extensive classical and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations are used to establish the structural features of the O state in bacteriorhodopsin (bR) and its conversion back to the bR ground state. The computed free energy surface is consistent with available experimental data for the kinetics and thermodynamics of the O to bR transition. The simulation results highlight the importance of the proton release group (PRG, consisting of Glu194/204) and the conserved arginine 82 in modulating the hydration level of the protein cavity. In particular, in the O state, deprotonation of the PRG and downward rotation of Arg82 lead to elevated hydration level and a continuous water network that connects the PRG to the protonated Asp85. Proton exchange through this water network is shown by ∼0.1-μs semiempirical QM/MM free energy simulations to occur through the generation and propagation of a proton hole, which is relayed by Asp212 and stabilized by Arg82. This mechanism provides an explanation for the observation that the D85S mutant of bacteriorhodopsin pumps chloride ions. The electrostatics-hydration coupling mechanism and the involvement of all titration states of water are likely applicable to many biomolecules involved in bioenergetic transduction.

Stereodynamic Imaging of Bromine Atomic Photofragments Eliminated from 1-Bromo-2-methylbutane Oriented via Hexapole State Selector.

Both single-laser and two-laser experiments were conducted to look into the ion-imaging of Br*(2P1/2) and Br(2P3/2) photofragmented from 1-bromo-2-methylbutane in the range 232-240 nm via a detection scheme of (2+1) resonance-enhanced multiphoton ionization. The angular analysis of these photofragment distributions yields the anisotropy parameter β = 1.88 ± 0.06 for the Br* excited state which arises from a parallel transition, while β = 0.63 ± 0.09 for the Br ground state indicates the contribution from both a perpendicular transition and a non-adiabatic transition. When a hexapole coupled with an orienting field was implemented, the parent molecules are spatially oriented to yield an orientation efficiency |⟨cosθ⟩| of 0.15. Besides the χ angle between the recoil velocity v and the transition dipole moment μ, orienting molecules allows for the evaluation of the angle α between v and the permanent molecular dipole moment d. The angular analysis of Br* photofragment distribution yields χ = 11.5° and α in the range from 160° to 180° with weak dependency. In the two-laser experiments, the angular anisotropy of Br photofragment distribution was found to be smaller (0.38 ± 0.10) when the photolysis wavelength was red-shifted to 240 nm, suggesting the increasing contributions from perpendicular transitions.

Function of Tyr185 in Stabilizing the Isomerization Equilibrium of the Retinal Chromophore in the Bacteriorhodopsin Ground State

Bacteriorhodopsin (bR), a member of the microbial rhodopsin family's seven trans-membrane receptors, functions as a light-driven proton pump for light energy capture and conversion in Halobacterium Salinarum. In the dark-adapted (DA) state, bR contains two thermally interconvertible retinal isomers: 13-cis, 15-syn retinal (bRcis), and all-trans, 15-anti retinal (bRtrans) with a molar ratio of 6:4 in the purple membrane. Absorption of a photon causes photoisomerization of the chromophore from the all-trans to the 13-cis, 15-anti configuration and triggers a series of structural rearrangements of the protein that initiates a vectorial translocation of a proton out of the cell.The function of Tyrosine 185 (Tyr185) in bR photoreaction mechanism has long been a topic of discussion, and been suggested to play an important role in stabilizing the pentagonal hydrogen-bonded network on the extracellular side of the protonated Schiff base during the photoreaction. However, little is to know on coupling of Tyr185 with the protonated Schiff base with respect to the thermal isomerization of the retinal chromophore at the bR ground state. Heteronuclear single molecular manipulation, combined with homonuclear correlation experiments, UV spectroscopy, light-induced transient absorption change measurements, molecular dynamic simulations and mutagenesis, has been established as a powerful tool for providing insight into the conformation and coupling of Tyr185 with the retinal chromophore at the bR ground state in the purple membrane. Our results show that the retinal chromophore in 13-cis and 15-syn configurations has more accumulated charge and, is more twisted and rigid than that in the all-trans configuration. Moreover, the cis-trans thermal isomerization equilibrium with the molar ratio of 6:4 at the ground state is stabilized by coupling with Tyr185.

Conformation Changes of a 7TM Receptor Caused by the Sample Environment as Studied by Multidisciplinary Biophysical Methods

Choosing an appropriate sample environment is critical in structural biology, not only for trapping functionally relevant intermediate states of a membrane protein, but also for interrogation of structure, conformation and dynamics to elucidate structure-function relationships. Functional assays and structural studies should therefore be performed in the same environment.Bacteriorhodopsin (bR), a member of the microbial rhodopsin family 7TM proteins, acting as a light-driven proton pump for light energy capture in Halobacterium Salinarum, has been studied by multidisciplinary approaches to reveal the molecular machinery of proton transfer and photocycle. For example, in order to study the functionally related structural change in M-state intermediate, different media with a high pH value have been used to extend the lifetime of M state. However, not much attention have been paid to the conformational changes that occur in the ground state, especially around the retinal binding pocket, due to local environment.Here, the conformation changes to the bR ground state, especially around the retinal binding pocket, have been studied by solid-state NMR through chemical shifts and torsion angle measurements, combined with the light-induced kinetic and UV spectroscopies. All the experimental results have been discussed in the context of X-ray crystal structures, and possible mechanisms have been proposed.

Characterization of vibrational wave packets by core-level high-harmonic transient absorption spectroscopy

Author(s): Hosler, ER; Leone, SR | Abstract: The ground-state vibrational wave packet produced by strong-field ionization of Br2 is characterized by femtosecond high-harmonic transient absorption spectroscopy. Vibrational motion is observed in time by a change in the 3d core-level transition energy with bond length 0.14 eV/pm to higher energies at shorter bond lengths. The wave packet has the expected period of 104 fs for the predominant v0v1 vibrational quantum beat, which is prepared near the outer turning point with a phase of 0.21π±0.05π due to the preferential ionization at short bond lengths. Simultaneous observation of a wave packet on the Br2+2Π g,3/2 ground state, prepared at the equilibrium bond distance of the ion, confirms preferential ionization near the inner turning point of the neutral. The Br2 ground-state wave packet has a degree of coherence ranging between 0.19 and 0.24, where unity is perfect coherence. The results utilize the sensitivity of core-level transient absorption spectroscopy to bond length and charge state, providing a means to analyze the formation and evolution of vibrational wave packets. © 2013 American Physical Society.

Plasmonic Field Enhancement of the Bacteriorhodopsin Photocurrent during Its Proton Pump Photocycle

The proton pump photocycle of bacteriorhodopsin (bR) produces photocurrent on a microsecond time scale which is assigned to the deprotonation step forming the M(412) intermediate. The return of the M(412) intermediate to the bR ground state (bR(570)) has two pathways: (1) thermally via multiple intermediates (which takes 15 ms) or (2) by a more rapid and direct process by absorbing blue light (which takes hundreds of nanoseconds). By using nanoparticles (Ag, Ag-Au, and Au NPs) having different surface plasmon resonance extinction spectra, it is found that Ag NPs whose spectrum overlaps best with the M(412) absorption regions enhance the stationary photocurrent 15 times. This large enhancement is proposed to be due to the accelerated photoexcitation rate of the M(412) (in the presence of the plasmon field of the light in this region) as well as short-circuiting of the photocycle, increasing its duty cycles.

Excitation of the M intermediates of wild-type bacteriorhodopsin and mutant D96N: temperature dependence of absorbance, electric responses and proton movements

The simplest proton pump known in biological systems, bacteriorhodopsin (bR), is the first ion-transporting membrane protein, the function of which can be described at the atomic level, with the aid of molecular dynamics calculations. To get additional experimental support for the proposed atomic level description of the function of bR, we studied a quasi-stable state of the protein molecule, the so-called M intermediate that plays a crucial role in the proton pumping process. The temperature dependence of the light-induced events occurring in the photocycle of wild-type bacteriorhodopsin and its mutant D96N were followed in detail. Absorbance changes, electric signals generated by charge motion inside the protein, and movement of protons in the protein solution interface either forward (proton release due to excitation of bR) or backward (uptake of protons due to the M excitation: “back-take”) were monitored. The obtained Arrhenius parameters indicate that the proton back-take is triggered by charge rearrangements in the protein similar to the proton release triggered by those during the L → M transition. The time necessary for proton back-take determines the reconstitution time of the bR ground state. The data are expected to be used in theoretical modeling of the bR function. Based on these results, a more detailed photocycle model is established to describe the proton pumping mechanism, implying a formal principle ("domino model") that is expected to hold also for other charge transfer proteins.

The Bacteriorhodopsin Carboxyl-Terminus Contributes to Proton Recruitment and Protein Stability

We examined functional and structural roles for the bacteriorhodopsin (bR) carboxyl-terminus. The extramembranous and intracellular carboxyl-terminus was deleted by insertion of premature translation stop codons. Deletion of the carboxyl-terminus had no effect on purple membrane (PM) lattice dimensions, sheet size, or the electrogenic environment of the ground-state chromophore. Removal of the distal half of the carboxyl-terminus had no effect on light-activated proton pumping, however, truncation of the entire carboxyl-terminus accelerated the rates of M-state decay and proton uptake approximately 3.7-fold and severely distorted the kinetics of proton uptake. Differential scanning calorimetry (DSC) and SDS denaturation demonstrated that removal of the carboxyl-terminus decreased protein stability. The DSC melting temperature was lowered by 6 degrees C and the calorimetric enthalpy reduced by 50% following removal of the carboxyl-terminus. Over the time range of milliseconds to hours at least 3 phases were required to describe the SDS denaturation kinetics for each bR construction. The fastest phases were indistinguishable for all bR's, and reflected PM solubilization. At pH 7.4, 20 degrees C, and in 0.3% SDS (w/v) the half-times of bR denaturation were 19.2 min for the wild-type, 12.0 min for the half-truncation and 3.6 min for the full-truncation. Taken together the results of this study suggest that the bR ground state exhibits two "domains" of stability: (1) a core chromophore binding pocket domain that is insensitive to carboxyl-terminal interactions and (2) the surrounding helical bundle whose contributions to protein stability and proton pumping are influenced by long-range interactions with the extramembranous carboxyl-terminus.

Active Internal Waters in the Bacteriorhodopsin Photocycle. A Comparative Study of the L and M Intermediates at Room and Cryogenic Temperatures by Infrared Spectroscopy

We present time-resolved room-temperature infrared difference spectra for the bacteriorhodopsin (bR) photocycle at 8 cm (-1) spectral and 5 micros temporal resolution, from 4000 to 800 cm (-1). An in situ hydration method allowed for a controlled and stable sample hydration (92% relative humidity), largely improving the quality of the data without affecting the functionality of bR. Experiments in both H 2 (16)O and H 2 (18)O were conducted to assign bands to internal water molecules. Room-temperature difference spectra of the L and M intermediates minus the bR ground state (L-BR and M-BR, respectively) were comprehensively compared with their low-temperature counterparts. The room-temperature M-BR spectrum was almost identical to that obtained at 230 K, except for a continuum band. The continuum band contains water vibrations from this spectral comparison between H 2 (16)O and H 2 (18)O, and no continuum band at 230 K suggests that the protein/solvent dynamics are insufficient for deprotonation of the water cluster. On the other hand, an intense positive broadband in the low-temperature L-BR spectrum (170 K) assigned to the formation of a water cavity in the cytoplasmic domain is absent at room temperature. This water cavity, proposed to be an essential feature for the formation of L, seems now to be a low-temperature artifact caused by restricted protein dynamics at 170 K. The observed differences between low- and room-temperature FTIR spectra are further discussed in light of previously reported dynamic transitions in bR. Finally, we show that the kinetics of the transient heat relaxation of bR after photoexcitation proceeds as a thermal diffusion process, uncorrelated with the photocycle itself.

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.

Time-Resolved Linear Dichroism and Linear Birefringence of Bacteriorhodopsin at Alkaline pH: Identification of Two N Substates with Different Orientations of the Transition Dipole Moment

Transient linear dichroism and linear birefringence changes in the photocycle of bacteriorhodopsin at alkaline pH were measured with magnetically oriented purple membrane samples using the method of isotropic excitation (Borucki, B.; Otto, H.; Heyn, M. P. J. Phys. Chem. B 1999, 103, 6371−6383). At pH 10.4, the accumulation of the O intermediate is negligible, and N is the only intermediate that is relevant in the M decay and the recovery of the bR ground state. We introduced a new analytical approach that is model-independent and makes use of the transient linear dichroism in combination with isotropic absorbance changes. SVD analysis reveals that only one species (spectral component), namely, the N intermediate, is involved in the M decay at pH 10.4. The spectrum of this intermediate and its average anisotropy are determined from an eigenvalue equation. The transient linear dichroism data suggest a transition between two substates of the N intermediate with different anisotropies (i.e., with different orientations of the electronic transition dipole moment). Assuming that the polar angle of the transition dipole moment with respect to the membrane normal is 70° in the bR ground state, we get 66.3° (upper limit) for the first and 67.9° (lower limit) for the second N substate. The reorientation of the chromophore in the N1 → N2 transition is probably associated with the movement of the protonated Schiff base away from Asp96, suggesting that it serves as a reprotonation switch for Asp96 by stabilizing the protonation state of the Schiff base. In contrast to measurements at neutral pH, the linear birefringence changes at alkaline pH contain a wavelength-independent component that is not due to the transient absorption changes and whose time dependence is correlated with the kinetics of the N intermediate. This result is attributed to an electro-optical effect caused by the large electric field generated by the transient negative charge on Asp96 in the N intermediate. Alternatively this change in Δn may be due to the major conformational change in the cytoplasmic region of the membrane during N.

Comparison of the Dynamics of the Primary Events of Bacteriorhodopsin in Its Trimeric and Monomeric States

In this paper, femtosecond pump-probe spectroscopy in the visible region of the spectrum has been used to examine the ultrafast dynamics of the retinal excited state in both the native trimeric state and the monomeric state of bacteriorhodopsin (bR). It is found that the excited state lifetime (probed at 490 nm) increases only slightly upon the monomerization of bR. No significant kinetic difference is observed in the recovery process of the bR ground state probed at 570 nm nor in the fluorescent state observed at 850 nm. However, an increase in the relative amplitude of the slow component of bR excited state decay is observed in the monomer, which is due to the increase in the concentration of the 13- cis retinal isomer in the ground state of the light-adapted bR monomer. Our data indicate that when the protein packing around the retinal is changed upon bR monomerization, there is only a subtle change in the retinal potential surface, which is dependent on the charge distribution and the dipoles within the retinal-binding cavity. In addition, our results show that 40% of the excited state bR molecules return to the ground state on three different time scales: one-half-picosecond component during the relaxation of the excited state and the formation of the J intermediate, a 3-ps component as the J changes to the K intermediate where retinal photoisomerization occurs, and a subnanosecond component during the photocycle.

The intensity dependent refractive index change of bacteriorhodopsin measured by the Z-scan and phase-modulated beams techniques

Abstract A comparative analysis of intensity dependent refractive index change of a thin polymer bacteriorhodopsin (bR) film by the Z -scan and phase-modulated beams techniques is given. The analysis is conducted at two excitation wavelengths, λ =543 and 633 nm, corresponding to the opposite slopes of the bR ground-state absorption band resulting in opposite signs of the refractive index change. A three-level model of the bR photo-cycle is proposed to describe the experimental data.

Kinetics of photo-active bacteriorhodopsin analog 3,4-didehydroretinal

Kinetics of the photo-induced processes of the transient states of the 3,4-didehydroretinal (3,4-dhr) modified bacteriorhodopsin (bR) was studied by a flash photolysis method in a water suspension at room temperature. The excitation initiated a photocycle with several transient intermediates similar to the trans photocycle of native bR. The main observation of the study was that although major part (80%) of the population of the M state relaxed via the O intermediate as in natural bR, 20% relaxed directly to the bR ground state in 200 ms.

Proton Transfer in Bacteriorhodopsin: Structure, Excitation, IR Spectra, and Potential Energy Surface Analyses by an ab Initio QM/MM Method

The structures, the optical properties, and the proton-transfer mechanism of bacteriorhodopsin (bR) are investigated by using an ab initio QM/MM calculation. The nature of the hydrogen bond network (HBN) formed in the retinal pocket is examined. The analysis is made for the “environmental” effect from the molecules involved in this HBN on the geometry and the excitation spectrum of retinal. It is found that IR spectra of the O−D stretching of the bound water molecules and the N−D stretching of the Schiff base retinal are very sensitive to the vibration−electron coupling among these molecules in the pocket. The hypothetical proton-transfer reaction path in the bR ground state is investigated, and the “environmental effect” on it is analyzed. The present calculation reveals that electronic interactions such as charge transfer and polarization, not included in most empirical methods, are very important to describe these properties of bR, the structures of HBN, the IR activities, and the proton-transfer mecha...

Light-induced denaturation of bacteriorhodopsin solubilized by octyl-beta-glucoside.

The structural stability of bacteriorhodopsin (bR) solubilized by octyl-beta-glucoside was studied by measuring the denaturation kinetics under visible light irradiation and in the dark. The denaturation of bR solubilized by 50 mM octyl-beta-glucoside was very slow at room temperature when it was left in the dark. However, its spontaneous denaturation was accelerated when the solubilized bR was irradiated by visible light. The denaturation kinetics under visible light irradiation and in the dark could be well described by a single decay constant. The activation energy for the denaturation of bR was estimated from the temperature dependence of decay time constants. The activation energy under visible light irradiation was 12.5 kcal/mol, which was much smaller than the corresponding value in the dark, 26.2 kcal/mol. These results strongly suggest that some of the photointermediate states are less stable than the ground state of bR. The critical temperature and the activation energy for denaturation of bR in the solubilized state were much lower than those in the 2D crystalline state. Comparing the denaturation behavior in the 2D crystalline state and that in the octyl-beta-glucoside-solubilized state, our findings suggest that protein-protein interaction contributes to the stability of this protein.

Fourier transform infrared double-flash experiments resolve bacteriorhodopsin's M1 to M2 transition

The orientation of the central proton-binding site, the protonated Schiff base, away from the proton release side to the proton uptake side is crucial for the directionality of the proton pump bacteriorhodopsin. It has been proposed that this movement, called the reprotonation switch, takes place in the M1 to M2 transition. To resolve the molecular events in this M1 to M2 transition, we performed double-flash experiments. In these experiments a first pulse initiates the photocycle and a second pulse selectively drives bR molecules in the M intermediate back into the BR ground state. For short delay times between initiating and resetting pulses, most of the M molecules being reset are in the M1 intermediate, and for longer delay times most of the reset M molecules are in the M2 intermediate. The BR-M1 and BR-M2 difference spectra are monitored with nanosecond step-scan Fourier transform infrared spectroscopy. Because the Schiff base reprotonation rate is kM1 = 0.8 x 10(7) s(-1) in the light-induced M1 back-reaction and kM2 = 0.36 x 10(7) s(-1) in the M2 back-reaction, the two different M intermediates represent two different proton accessibility configurations of the Schiff base. The results show only a minute movement of one or two peptide bonds in the M1 to M2 transition that changes the interaction of the Schiff base with Y185. This backbone movement is distinct from the larger one in the subsequent M to N transition. No evidence of a chromophore isomerization is seen in the M1 to M2 transition. Furthermore, the results show time-resolved reprotonation of the Schiff base from D85 in the M photo-back-reaction, instead of from D96, as in the conventional cycle.

Spin-labeling studies of the conformational changes in the vicinity of D36, D38, T46, and E161 of bacteriorhodopsin during the photocycle

Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin-labeled bacteriorhodopsin mutants is used to study structural changes during the photocycle. After exchange of the native amino acids D36 and D38 in the A-B loop, E161 in the E-F loop, and T46 in the putative proton channel by cysteines, these positions were modified by a methanethiosulfonate spin label. Time-resolved EPR spectroscopy reveals spectral changes during the photocycle for the mutants with spin labels attached to C36, C161, and C46. A comparison of the transient spectral amplitudes with simulated EPR difference spectra shows that the detected signals are due to changes in the spin label mobility and not to possible polarity changes in the vicinity of the attached spin label. The kinetic analysis of the EPR and the visible data with a global fitting procedure exhibits a structural rearrangement near position 161 in the E-F loop in the M state. The environmental changes at positions 36 and 46, however, occur during the M-to-N transition. All structural changes reverse with the recovery of the BR ground state. No structural changes are detected with a spin label attached to C38.

High-resolution (2 + 1) multiphoton ionization spectroscopy of Br 2

High-resolution (2 + 1) multiphoton ionization spectroscopy of Br 2 was studied. Rotationally resolved spectra of the gerade Rydberg state [Π 3 2 ]4d(υ′ = 1, 2) are reported for the first time. Rotational constants B υ′ were deduced for the three isotopes, which are quite close to the data of the ground state of Br + 2. In addition, the angular momentum Ω of the state was suggested to be reassigned to be 0.