Studies on pyrylretinal analogues of bacteriorhodopsin

The retinal analogues 3-methyl-5-(1-pyryl)-2E,4E-pentadienal (1) and 3,7-dimethyl-9-(1-pyryl)-2E,4E,6E,8E-nonatetr aenal (2), which contain the tetra aromatic pyryl system, have been synthesized and characterized in order to examine the effect of the extended ring system on the binding capabilities and the function of bacteriorhodopsin (bR). The two bR mutants, E194Q and E204Q, known to have distinct proton-pumping patterns, were also examined so that the effect of the bulky ring system on the proton-pumping mechanism could be studied. Both retinals formed pigments with all three bacterioopsins, and these pigments were found to have absorption maxima in the range 498-516 nm. All the analogue pigments showed activity as proton pumps. The pigment formed from wild-type apoprotein bR with 1 (with the shortened polyene side chain) showed an M intermediate at 400 nm and exhibited fast proton release followed by proton uptake. Extending the polyene side chain to the length identical with retinal, analogue 2 with wild-type apoprotein gave a pigment that shows M and O intermediates at 435 nm and 650 nm, respectively. This pigment shows both fast and slow proton release at pH 7, suggesting that the pKa of the proton release group (in the M-state) is higher in this pigment compared to native bR. Hydrogen azide ions were found to accelerate the rise and decay of the O intermediate at neutral pH in pyryl 2 pigment. The pigments formed between 2 and E194Q and E204Q showed proton-pumping behavior similar to pigments formed with the native retinal, suggesting that the size of the chromophore ring does not alter the protein conformation at these sites.     

Excitation of the M Intermediates of Bacteriorhodopsin†

Protein electric response signals (PERS) of the M intermediates of wild‐type bacteriorhodopsin (bR) were recorded. Contrary to earlier findings reporting on a single‐phase response upon excitation of the M intermediates, a kinetic analysis of the signals revealed the existence of three components, the fastest and the slowest ones of negative, while the middle one of positive sign with respect to the normal direction of proton pumping. Based on proton motion indicator experiments and molecular dipole calculations, the components were assigned to proton transfer steps and conformational changes driving the bR molecule back from the M to the ground state upon blue light excitation. The fastest, negative pump component was assigned to the proton transfer from D85 to the Schiff base. The subsequent positive component was attributed to rearrangements in the protein core (in the vicinity of the retinal molecule), triggered by the primary proton transfer process. The slowest component was established to reflect charge rearrangements associated with proton uptake by the protein from the bulk.     

Photocurrent of purple membrane adsorbed onto a thin polymer film: action characteristics of the local anesthetics

The proton pumping activity of bacteriorhodopsin (bR) in the purple membrane adsorbed onto a thin polymer film as a solid support for electrical measurements has been examined in the presence of local anesthetics and 1-alcohols as an anesthetic model. This membrane adsorbed system provided high reproducibility of the photocurrents in bR due to the mechanical and the chemical stability and the electric properties of the thin polymer film. As the concentrations of the local anesthetics increased, the photocurrents generated by the proton pump of bR were cooperatively suppressed and the changes in the photocurrents were reversible. From the dose–response curves for the anesthetics, the concentration (EC₅₀) required for a 50% suppression showed a marked specificity in the order of lidocaine>bupivacaine>tetracaine>dibucaine. The suppression of the photocurrent in bR was more effective for the uncharged form of the local anesthetics than for the charged one. The absorption and fluorescence spectra suggested that the charged form of the anesthetics was bound to the purple membrane surface, while their uncharged form interacted with the hydrophobic portions of the purple membrane interior rather than with the membrane surface. From the dose–response curves for the 1-alcohols, an increase in hydrophobicity in their molecules effectively suppressed the photocurrent of bR. We found that the binding of hydrophobic organic cations such as tetracaine hydrochloride and bupivacaine hydrochloride to the blue membrane with loss of the proton pump, which was prepared by removal of the cations from the purple membrane, could regenerate the proton pumping activity. The photocurrent in bR in the purple membrane adsorbed onto a thin solid film sensitively responded to different local anesthetics.     

PROTON TRANSLOCATION and CONFORMATIONAL CHANGES DURING THE BACTERIORHODOPSIN PHOTOCYCLE: TIME-RESOLVED STUDIES WITH MEMBRANE-BOUND OPTICAL PROBES and X-RAY DIFFRACTION*

Abstract– For the first time, we monitored time-resolved conformational changes inherent in bacterio-rhodopsin's reaction cycle as well as the H⁺-release events directly at the surface of bacteriorhodopsin (bR) at room temperature. Signals of optical pH-indicators in the aqueous bulk phase were compared with those of probes covalently linked to bR. The kinetics of H⁺-translocation and the correlation of the photocycle with the pumping cycle can only be determined with indicators bound to bR. The proton appears at the extracellular surface during the L₅₅₀ to M₄₁₂ transition. Upon short photo-excitation, diffraction patterns were obtained from both guanidine hydrochloride-treated wild type bR and an Asp96Asn mutant with millisecond time resolution by x-ray synchrotron radiation. The measured time course and location of the structural changes confirm and extend our previous static neutron diffraction study at -180°C. The temporal correlation of photocycle intermediates and proton translocation steps with structural changes in the protein under similar environmental conditions strongly contribute to the understanding of the pumping mechanism.     

Key role of active-site water molecules in bacteriorhodopsin proton-transfer reactions

The functional mechanism of the light-driven proton pump protein bacteriorhodopsin depends on the location of water molecules in the active site at various stages of the photocycle and on their roles in the proton-transfer steps. Here, free energy computations indicate that electrostatic interactions favor the presence of a cytoplasmic-side water molecule hydrogen bonding to the retinal Schiff base in the state preceding proton transfer from the retinal Schiff base to Asp85. However, the nonequilibrium nature of the pumping process means that the probability of occupancy of a water molecule in a given site depends both on the free energies of insertion of the water molecule in this and other sites during the preceding photocycle steps and on the kinetic accessibility of these sites on the time scale of the reaction steps. The presence of the cytoplasmic-side water molecule has a dramatic effect on the mechanism of proton transfer: the proton is channeled on the Thr89 side of the retinal, whereas the transfer on the Asp212 side is hindered. Reaction-path simulations and molecular dynamics simulations indicate that the presence of the cytoplasmic-side water molecule permits a low-energy bacteriorhodopsin conformer in which the water molecule bridges the twisted retinal Schiff base and the proton acceptor Asp85. From this low-energy conformer, proton transfer occurs via a concerted mechanism in which the water molecule participates as an intermediate proton carrier.     

Quantum dynamics of the femtosecond photoisomerization of retinal in bacteriorhodopsin

The membrane protein bacteriorhodopsin contains all-trans-retinal in a binding site lined by amino acid side groups and water molecules that guide the photodynamics of retinal. Upon absorption of light, retinal undergoes a subpicosecond all-trans-->13-cis phototransformation involving torsion around a double bond. The main reaction product triggers later events in the protein that induce pumping of a proton through bacteriorhodopsin. Quantum-chemical calculations suggest that three coupled electronic states, the ground state and two closely lying excited states, are involved in the motion along the torsional reaction coordinate phi. The evolution of the protein-retinal system on these three electronic surfaces has been modelled using the multiple spawning method for non-adiabatic dynamics. We find that, although most of the population transfer occurs on a timescale of 300 fs, some population transfer occurs on a longer timescale, occasionally extending well beyond 1 ps.     

A NOVEL BACTERIORHODOPSIN ANALOGUE WITH CONFORMATIONALLY 6-s-cis FIXED RETINALS

Abstract— The artificial pigments of bacteriorhodopsin (bR) were synthesized with retinal analogues which have 6-s-cis fixed conformation. In order to study the shape of binding pocket around β-ionone ring of retinal, we used three different 6-s-cis fixed retinals (1–3), which have a gem-dimethyl group at a different position of the ring structure. These three 6-s-cis fixed retinals produced bR analogue pigments with absorption maxima at 574, 594 and 584 nm, respectively. These analogue pigments showed proton pumping activities upon incorporation into vesicles, indicating that 6-s-trans structure of retinal is not necessary to pump protons. Although these retinal analogues possess the same conjugated π-electron systems, the wavelengths of maximum absorbance of the analogue pigments were different from each other, indicating a distinctly steric interaction between retinal ring portion and apoprotein. The analogue pigments were also different from each other in regeneration rate and the number of the final products. These differences resulted from a difference in the position of the dimethyl substituent at the ring structure. Based on the results the molecular structure around β-ionone ring of retinal-binding pocket was discussed.     

Combined QM/MM study of the opsin shift in bacteriorhodopsin

Combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations of bacteriorhodopsin (bR) in the membrane matrix have been carried out to determine the factors that make significant contributions to the opsin shift. We found that both solvation and interactions with the protein significantly shifts the absorption maximum of the retinal protonated Schiff base, but the effects are much more pronounced in polar solvents such as methanol, acetonitrile, and water than in the protein environment. The differential solvatochromic shifts of PSB in methanol and in bR leads to a bathochromic shift of about 1800 cm(-1). Because the combined QM/MM configuration interaction calculation is essentially a point charge model, this contribution is attributed to the extended point-charge model of Honig and Nakanishi. The incorporation of retinal in bR is accompanied by a change in retinal conformation from the 6-s-cis form in solution to the 6-s-trans configuration in bR. The extension of the pi-conjugated system further increases the red-shift by 2400 cm(-1). The remaining factors are due to the change in dispersion interactions. Using an estimate of about 1000 cm(-1) in the dispersion contribution by Houjou et al., we obtained a theoretical opsin shift of 5200 cm(-1) in bR, which is in excellent agreement with the experimental value of 5100 cm(-1). Structural analysis of the PSB binding site revealed the specific interactions that make contributions to the observed opsin shift. The combined QM/MM method used in the present study provides an opportunity to accurately model the photoisomerization and proton transfer reactions in bR.     

Dissection of Environmental Changes at the Cytoplasmic Surface of Light‐activated Bacteriorhodopsin and Visual Rhodopsin: Sequence of Spectrally Silent Steps†

The physico‐chemical properties as well as the conformation of the cytoplasmic surface of the 7‐helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100 , 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light‐activated bR and rhodopsin detected by time‐resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real‐time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin‐II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time‐resolved fluorescence anisotropy experiments reveal an increase in steric restriction of loop flexibility because of changes in the surrounding protein environment in both the M‐intermediate as well as the active metarhodopsin‐II state. The kinetics of the fluorescence changes at the rhodopsin surface uncover multiple transitions, suggesting metarhodopsin‐II substates with different surface properties. Proton uptake from the aqueous bulk phase correlates with the first transition, while late proton release seems to parallel the second transition. The last transition between states of different surface properties correlates with metarhodopsin‐II decay.     

The proton uptake channel of bacteriorhodopsin as studied by a photoelectrochemical method

A series of the mutant proteins (D96N, D96N/D85N, D115N, L93T, T46V, V49A) where the residues are located at the cytoplasmic domain of bacteriorhodopsin (bR) were studied photoelectrochemically and their photocurrent response characteristics at the electrode/electrolyte interface were compared with those of the wild-type bR. While the wild-type bR of normal proton pumping activity yields symmetrical cathodic (positive) and anodic (negative) responses, corresponding to proton release and proton uptake, respectively, these mutants, with the exception of D115N, showed diminished amplitudes in the negative response. This indicates retardation of proton translocation from the cytoplasmic surface to the retinal Schiff base. The mutation that gave the strongest influence on the negative response was D96N while moderate influence was obtained with L93T, T46V, and V49A. These results suggest that residues other than D96 also participate in the cytoplasmic proton uptake channel, either by interacting with D96 directly or by forming a hydrogen-bonded network with water molecules. The D96N/D85N double mutant yielded little response at neutral pH, but the response was partially recovered by addition of azide, while it was fully recovered in the single mutant D96N. The D115N mutant showed the response profile that closely resembles the wild-type, indicating that D115 is not crucially involved in the event of proton transfer relay at the cytoplasmic region. It was also found that every mutant in this study releases protons prior to uptake at the other membrane surface, as does the wild-type.     

Temperature-dependent solid-state electron transport through bacteriorhodopsin: experimental evidence for multiple transport paths through proteins

Electron transport (ETp) across bacteriorhodopsin (bR), a natural proton pump protein, in the solid state (dry) monolayer configuration, was studied as a function of temperature. Transport changes from thermally activated at T > 200 K to temperature independent at <130 K, similar to what we have observed earlier for BSA and apo-azurin. The relatively large activation energy and high temperature stability leads to conditions where bR transports remarkably high current densities above room temperature. Severing the chemical bond between the protein and the retinal polyene only slightly affected the main electron transport via bR. Another thermally activated transport path opens upon retinal oxime production, instead of or in addition to the natural retinal. Transport through either or both of these paths occurs on a background of a general temperature-independent transport. These results lead us to propose a generalized mechanism for ETp across proteins, in which tunneling and hopping coexist and dominate in different temperature regimes.     

Role of hydrogen-bond network in energy storage of bacteriorhodopsin's light-driven proton pump revealed by ab initio normal-mode analysis

Vibrational modes of the hydrogen-bond network in the binding site of bacteriorhodopsin (bR), a protein in halobacteria functioning as a light-driven proton pump, were investigated by an ab initio quantum mechanical/molecular mechanical (QM/MM) method. Normal-mode analysis calculations for O-D and N-D stretching modes of internal water molecules and the Schiff base of the retinal chromophore in the early intermediate state, K, reproduced well experimentally observed vibrational spectra. Supported by agreement with observed spectra, the QM/MM calculation suggests that weakened hydrogen bonds upon photoisomerization of the chromophore are an important means of energy storage in bR.     

Participation of the BC Loop in the Correct Folding of Bacteriorhodopsin as Revealed by Solid‐state NMR†

Structural changes in bacteriorhodopsin (bR) in two different processes of retinal reconstitutions were investigated by observing the ¹³C and ¹⁵N solid‐state NMR spectra of [1‐¹³C]Val‐ and [¹⁵N]Pro‐labeled bR. We found that NMR signals of the BC loop were sensitive to changes in protein structure and dynamics, from wild‐type (WT) bR to bacterio‐opsin (bO), regenerated bR and E1001 bR. Regenerated bR was prepared following the addition of retinal into bO obtained from photobleached WT‐bR. E1001 bR was cultured from a retinal‐deficient strain termed E1001 following the addition of retinal to growing cells. ¹⁵N NMR signal at Pro70 in the BC loop in WT‐bR was observed at 122.4 p.p.m., whereas signals were not apparent or partly suppressed in bO and regenerated bR, respectively. Similarly, the ¹³C NMR signal at Val69 in the BC loop at 172.0 p.p.m. that was observed in WT‐bR was significantly decreased in both regenerated bR and bO. These results suggest that the dynamic structure of the BC loop in bO was substantially altered following the removal of retinal. As a consequence, the correct protein structure failed to be recovered via the regenerating process of retinal to bO. On the other hand, ¹³C and ¹⁵N NMR signals at the BC loop in E1001 bR appeared at positions identical to those of WT‐bR. The results of the current study indicate that the BC loop may not always fold correctly in the regenerated bR, which leads to different properties in the regenerated bR compared to that of WT‐bR.     

Pressure-induced isomerization of retinal on bacteriorhodopsin as disclosed by fast magic angle spinning NMR

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light-driven proton pump. We have detected pressure-induced isomerization of retinal in bR by analyzing 15N cross polarization-magic angle spinning (CP-MAS) NMR spectra of [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) in protonated Schiff base at 148.0 and 155.0 ppm, respectively, at the MAS frequency of 4 kHz in the dark. When the MAS frequency was increased up to 12 kHz corresponding to the sample pressure of 63 bar, the 15N-NMR signals of [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-15N]Lys in Schiff base of 13-cis retinal at 155.0 ppm was observed when the MAS frequency was decreased from 12 to 4 kHz. These results showed that the equilibrium constant of [all-trans-bR]/[13-cis-bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen-bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all-trans to 13-cis state in the membrane protein bR.     

Bacteriorhodopsin O-state Photocycle Kinetics: A Surfactant Study

The spectroscopy and dynamics of interaction between the O intermediate of bacteriorhodopsin (bR) and several surfactants (cetrimonium bromide [CTAB], sodium dodecyl sulfate [SDS] and diethylene glycol mono- n -hexyl ether [C6E2]) were investigated using steady-state UV–VIS spectrometry, circular dichroism spectroscopy and time-resolved absorption techniques. The steady-state spectral results show that bR can retain its trimeric state without severe damage in the molar concentration ratio of C6E2/bR ranging up to 4000. Time-resolved observations indicate that the rise and decay rates and transient populations of the O state can be increased in the presence of nonionic surfactant C6E2; however, these studies indicate the opposite phenomenon in the presence of the ionic surfactants CTAB and SDS. The observed 40% enhancement in the transient population of the O intermediate state that results from treatment of C6E2 is proposed to result from an expanding bR structure, which leads to more effective proton pumping efficiency in the photosynthetic system of bR.     

Potential Proton‐Release Channels in Bacteriorhodopsin

The protein bacteriorhodopsin pumps protons across a bacterial membrane; its pumping cycle is triggered by the photoisomerization of a retinal cofactor and involves multiple proton‐transfer reactions between intermittent protonation sites. These transfers are either direct or mediated by hydrogen‐bonded networks, which may include internal water molecules. The terminal step of the proton‐transfer sequence is the proton release from a pocket near Glu194 and Glu204 to the extracellular bulk during the transition from the L to the M photointermediate states. The polar and charged side chains connecting these two regions in the crystal structures show no structural changes between the initial bR state and the L/M states, and no intermittent protonation changes have been detected so far in this region. Based on biomolecular simulations, we propose two potential proton‐release channels, which connect the release pocket to the extracellular medium. In simulations of the L photointermediate we observe bulk water entering these channels and forming transient hydrogen‐bonded networks, which could serve as fast deprotonation pathways from the release pocket to the bulk via a Grotthuss mechanism. For the first channel, we find that the triple Arg7, Glu9, and Tyr79 acts as a valve, thereby gating water uptake and release. The second channel has two release paths, which split at the position Asn76/Pro77 underneath the release group. Here, water molecules either exchange directly with the bulk or diffuse within the protein towards Arg 134/Lys129, where the exchange with the bulk occurs.     

pH DEPENDENCE OF THE ABSORPTION SPECTRA AND PHOTOCHEMICAL TRANSFORMATIONS OF THE ARCHAERHODOPSINS

Abstract —Two strains of archaebacteria have been found to contain light-driven proton pumping pigments analogous to bacteriorhodopsin (bR) in Halobacterium salinarium . These proteins are called archaerhodopsin-1 (aR-1) and archaerhodopsin-2 (aR-2). Their high degree of sequence identity with bR within the putative proton channel enables us to draw some conclusions about the roles of regions where differences in amino acids exist, and in particular the surface residues, on the structure and function of retinal-based proton pumps. We have characterized the spectral and photochemical properties of these two proteins and compared them to the corresponding properties of bR. While there are some differences in absorbance maxima and kinetics of the photocycle, most of the properties of aR-1 and aR-2 are similar to those of bR. The most striking differences of these proteins with bR are the lack of an alkaline-induced red-shifted absorption species and a dramatic (apparent) decrease in the light-induced transient proton release. In membrane sheet suspensions of aR-1 at 0.15 M KCI, the order of proton release and uptake appears opposite that of bR, in which proton release precedes uptake. The nature of this behavior appears to be due to differences in the amino acid sequence at the surfaces of the proteins. In particular, the residue corresponding to the lysine at position 129 of the extracellular loop region of bR is a histidine in aR-1 and could regulate the efficient release of protons into solution in bR.     

Proton storage site in bacteriorhodopsin: new insights from quantum mechanics/molecular mechanics simulations of microscopic pK(a) and infrared spectra

Identifying the group that acts as the proton storage/loading site is a challenging but important problem for understanding the mechanism of proton pumping in biomolecular proton pumps, such as bacteriorhodopsin (bR) and cytochrome c oxidase. Recent experimental studies of bR propelled the idea that the proton storage/release group (PRG) in bR is not an amino acid but a water cluster embedded in the protein. We argue that this idea is at odds with our knowledge of protein electrostatics, since invoking the water cluster as the PRG would require the protein to raise the pK(a) of a hydronium by almost 11 pK(a) units, which is difficult considering known cases of pK(a) shifts in proteins. Our recent quantum mechanics/molecular mechanics (QM/MM) simulations suggested an alternative "intermolecular proton bond" model in which the stored proton is shared between two conserved Glu residues (194 and 204). Here we show that this model leads to microscopic pK(a) values consistent with available experimental data and the functional requirement of a PRG. Extensive QM/MM simulations also show that, independent of a number of technical issues, such as the influence of QM region size, starting X-ray structure, and nuclear quantum effects, the "intermolecular proton bond" model is qualitatively consistent with available spectroscopic data. Potential of mean force calculations show explicitly that the stored proton strongly prefers the pair of Glu residues over the water cluster. The results and analyses help highlight the importance of considering protein electrostatics and provide arguments for why the "intermolecular proton bond" model is likely applicable to the PRG in biomolecular proton pumps in general.     

Color tuning in rhodopsins: the mechanism for the spectral shift between bacteriorhodopsin and sensory rhodopsin II

The mechanism of color tuning in the rhodopsin family of proteins has been studied by comparing the optical properties of the light-driven proton pump bacteriorhodopsin (bR) and the light detector sensory rhodopsin II (sRII). Despite a high structural similarity, the maximal absorption is blue-shifted from 568 nm in bR to 497 nm in sRII. The molecular mechanism of this shift is still a matter of debate, and its clarification sheds light onto the general mechanisms of color tuning in retinal proteins. The calculations employ a combined quantum mechanical/molecular mechanical (QM/MM) technique, using a DFT-based method for ground state properties and the semiempirical OM2/MRCI method and ab initio SORCI method for excited state calculations. The high efficiency of the methodology has allowed us to study a wide variety of aspects including dynamical effects. The absorption shift as well as various mutation experiments and vibrational properties have been successfully reproduced. Our results indicate that several sources contribute to the spectral shift between bR and sRII. The main factors are the counterion region at the extracellular side of retinal and the amino acid composition of the binding pocket. Our analysis allows a distinction and identification of the different effects in detail and leads to a clear picture of the mechanism of color tuning, which is in good agreement with available experimental data.