Structure, dynamics, and function of bacteriorhodopsin.
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Bacteriorhodopsins↗
Biomedical subjects
N A Dencher
Publications and source records attributed to N A Dencher.
Mechanism of proton entry into the cytoplasmic section of the proton-conducting channel of bacteriorhodopsin.
Bacteriorhodopsin is the light-driven proton-pumping protein of Halobacterium salinarum that extracts protons from the well-buffered cytoplasmic space within the time limits set by the photocycle turnover. The specific mechanism of the proton uptake by the cytoplasmic surface of the protein was investigated in this study by the laser-induced proton pulse technique. The purple membrane preparations were labeled by fluorescein at two residues (36 or 38) of the cytoplasmic surface of the protein, sites that are close to the orifice of the proton-conducting channel. The membranes were pulsed by protons discharged from photoexcited pyranine [Nachliel, E., Gutman, M., Kiryati, S., and Dencher, N.A. (1996) Proc. Nat Acad. Sci. U.S.A. 93, 10747-10752). The reaction of the discharged protons with the pyranine anion and the fluorescein was measured with sub-microsecond resolution. The experimental signals were reconstructed through numeric integration of differential rate equations which quantitated the rates of all proton transfer reactions between all reactants present in the system. The interaction of protons with the orifice of the cytoplasmic channel is enhanced by the exposed carboxylates of the protein. A cluster of three carboxylates acts as a strong proton attractor site while one carboxylate, identified as D36, acts as a mediator that delivers the proton to the channel. The combination of these reactions render the surface of the protein with properties of a proton-collecting antenna. The size of the collecting area is less than that of the protein's surface.
Bacteriorhodopsins↗
The tertiary structural changes in bacteriorhodopsin occur between M states: X-ray diffraction and Fourier transform infrared spectroscopy.
The tertiary structural changes occurring during the photocycle of bacteriorhodopsin (BR) are assigned by X-ray diffraction to distinct M states, M1 and M2. Purple membranes (PM) of the mutant Asp96Asn at 15, 57, 75 and 100% relative humidity (r.h.) were studied in a parallel X-ray diffraction and Fourier transform infrared (FTIR) spectroscopic investigation. Light-dependent conformational changes of BR-Asp96Asn are observed at high hydration levels (100 and 75% r.h.) but not in partially dehydrated samples (57 and 15% r.h.). The FTIR spectra of continuously illuminated samples at low and high hydration, despite some differences, are characteristic of the M intermediate. The changes in diffraction patterns of samples in the M2 state are of the same magnitude as those of wild-type samples trapped with GuaHCl in the M(G) state. Additional large changes in the amide bands of the FTIR spectra occur between M2 and M(G). This suggests, that the tertiary structural changes between M1 and M2 are responsible for the switch opening the cytoplasmic half-channel of BR for reprotonation to complete the catalytic cycle. These tertiary structural changes seem to be triggered by a charge redistribution which might be a common feature of retinal proteins also in signal transduction.
Amino Acid Sequence↗
Picosecond molecular motions in bacteriorhodopsin from neutron scattering.
The characteristics of internal molecular motions of bacteriorhodopsin in the purple membrane have been studied by quasielastic incoherent neutron scattering. Because of the quasihomogeneous distribution of hydrogen atoms in biological molecules, this technique enables one to study a wide variety of intramolecular motions, especially those occurring in the picosecond to nanosecond time scale. We performed measurements at different energy resolutions with samples at various hydration levels within a temperature range of 10-300 K. The analysis of the data revealed a dynamical transition at temperatures Td between 180 K and 220 K for all motions resolved at time scales ranging from 0.1 to a few hundred picoseconds. Whereas below Td the motions are purely vibrational, they are predominantly diffusive above Td, characterized by an enormously broad distribution of correlation times. The variation of the hydration level, on the other hand, mainly affects motions slower than a few picoseconds.
Bacteriorhodopsins↗
Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane.
The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.
Bacteriorhodopsins↗
Internal molecular motions of bacteriorhodopsin: hydration-induced flexibility studied by quasielastic incoherent neutron scattering using oriented purple membranes.
Quasielastic incoherent neutron scattering from hydrogen atoms, which are distributed nearly homogeneously in biological molecules, allows the investigation of diffusive motions occurring on the pico- to nanosecond time scale. A quasielastic incoherent neutron scattering study was performed on the integral membrane protein bacteriorhodopsin (BR), which is a light-driven proton pump in Halobacterium salinarium. BR is embedded in lipids, forming patches in the cell membrane of the organism, which are the so called purple membranes (PMs). Measurements were carried out at room temperature on oriented PM-stacks hydrated at two different levels (low hydration, h = 0.03 g of D2O per g of PM; high hydration, h = 0.28 g of D2O per g of PM) using time-of-flight spectrometers. From the measured spectra, different diffusive components were identified and analyzed with respect to the influence of hydration. This study supports the idea that a decrease in hydration results in an appreciable decrease in internal molecular flexibility of the protein structure. Because it is known from studies on the function of BR that the pump activity is reduced if the hydration level of the protein is insufficient, we conclude that the observed diffusive motions are essential for the function of this protein. A detailed analysis and classification of the different kinds of diffusive motions, predominantly occurring in PMs under physiological conditions, is presented.
Bacteriorhodopsins↗
Impaired plasticity of neurons in aging. Biochemical, biophysical, and behavioral studies.
Age-related correlation of impaired plasticity of neurons (biochemical and biophysical aspects) and behavioral alterations were investigated in young (3.5 months) and extremely aged (approximately 40 months) female Wistar rats. Age-dependent significant differences in second messenger (cAMP and Ins (1,4,5)P3) concentration and signal transduction via muscarinic and dopaminergic receptors were found. The results point to the specifically impaired coupling between dopamine D1 receptor and GS protein, which underlies normal brain aging. However, cholinergic neurotransmission may be modulated at another level in extremely aged rats. Thus, it appears that the site of affection in coupling of receptor and G protein and/or G protein-dependent signal transduction in aging cannot be generalized. This indicates that alterations in the coupling of signal transduction depend on diverse neurotransmitter receptors with advanced age. The age-dependent alterations in the cAMP and PI signal pathways could be due to changes in the physical properties of the membranes. To support this hypothesis, age-dependent changes in the physical state and the biochemical composition of synaptosomal membranes from the cortex, cerebellum, and striatum were examined by measuring the steady-state fluorescence amisotropy of the membrane probes 1,6-diphenyl-1,3,5-hexatriene (DPH), trimethylammonium-DPH (TMA-DPH), and trimethylammoniumpropyl-DPH (TMAP-DPH). Significant differences in the physical properties of the synaptosomal membranes existed between young and very aged rats, expressed by a higher anisotropy in the 40-month-old rat brain tissue. The changes in the physical properties of the membranes were in line with the determined age-dependent alterations in the chemical composition, e.g., the increase in cholesterol content of the aged membranes.
Age Factors↗
D38 is an essential part of the proton translocation pathway in bacteriorhodopsin.
At present, almost no knowledge exists about the functional relevance of the amino acid residues at the cytoplasmic (CP) surface of the light-driven proton pump bacteriorhodopsin (BR) although a prerequisite for efficient vectorial proton translocation is the efficient capture of protons from the alkaline cytoplasm of the cell. To identify residues involved in the proton transfer reaction steps in the CP part of BR, the aspartic and glutamic amino acids D36, D38, D102, D104, and E161 were replaced by cysteine and arginine (i.e., a negatively charged residue by a neutral or positive one at the pH of investigation). The effect of these replacements on the photo- and transport cycle was examined by time-resolved visible and infrared spectroscopy, biochemical modification studies, and activity assays in intact cells. Of the five CP amino acids studied, only the replacement of D38 resulted in severe alterations of the reaction steps in BR during the second half of the photocycle. Our data show that D38, which seemed to be a freely accessible CP surface residue lacking functional importance, is an essential part of the CP proton uptake pathway connecting the membrane surface with the Schiff base of BR, probably as the first amino acid residue at the CP entrance. D38 influences the late steps in the functional cycle, such as the occurrence of the intermediates N and O, the modulation of the hydrogen-network, the conformational changes in the protein moiety, and the deprotonation/reprotonation of D96. Opposed to this function, the surface-exposed amino acids D36, D102, D104, and E161 seem to efficiently collect protons from the aqueous bulk phase and funnel them to the entrance of the CP proton pathway.
Amino Acid Sequence↗
Light-induced isomerization causes an increase in the chromophore tilt in the M intermediate of bacteriorhodopsin: a neutron diffraction study.
Bacteriorhodopsin (BR) was regenerated with two selectively deuterated retinals, one with 11 deuterons in the beta-ionone ring (D11) and the other with 5 deuterons (D5) at the end of the polyene chain closest to the Schiff base at carbon atoms C-14, C-15, and C-20. Both label positions (centers of deuteration) were obtained from difference Fourier maps of projections onto the plane of the membrane by neutron diffraction at 90 K, both in the light-adapted ground-state BR568 and in the photocycle intermediate M412. To retard the decay of M412, purple membrane films were soaked in 0.1 M or 1 M guanidine hydrochloride at pH 9.6. M412 was produced by illuminating oriented membrane films at physiological temperature (278 K), followed by rapid cooling to 90 K in the absence of light. The results show that in the projected structure the ring position is unaltered during the transition from BR568 to M412, whereas the position of the D5 label shifts by 1.4 +/- 0.9 A toward the ring. The shortened interlabel distance in the projected structure for the M412 state implies that as a result of the all-trans/13-cis isomerization, the C-5 to C-13 part of the polyene chain tilts out of the plane of the membrane toward the cytoplasm by about 11 degrees +/- 6 degrees. Pairwise comparison of data sets with the same retinal for the two photocycle states M412 and BR568 leads to four difference-density maps for the protein, which are in agreement with previous work. They show changes in the protein density near helices G and F.
Bacteriorhodopsins↗
Proton migration along the membrane surface and retarded surface to bulk transfer.
Since the proposal of the chemiosmotic theory there has been a continuing debate about how protons that have been pumped across membranes reach another membrane protein that utilizes the established pH gradient. Evidence has been gathered in favour of a 'delocalized' theory, in which the pumped protons equilibrate with the aqueous bulk phase before being consumed, and a 'localized' one, in which protons move exclusively along the membrane surface. We report here that after proton release by an integral membrane protein, long-range proton transfer along the membrane surface is faster than proton exchange with the bulk water phase. The rate of lateral proton diffusion can be calculated by considering the buffer capacity of the membrane surface. Our results suggest that protons can efficiently diffuse along the membrane surface between a source and a sink (for example H(+)-ATP synthase) without dissipation losses into the aqueous bulk.
Arylsulfonates↗
Co-reconstitution of plasma membrane H(+)-ATPase from yeast and bacteriorhodopsin into liposomes. ATP hydrolysis as a function of external and internal pH.
The H(+)-ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The enzyme was reconstituted with bacteriorhodopsin into asolectin liposomes by detergent dialysis at a molar ratio of 1 H(+)-ATPase to 50 bacteriorhodopsins. The overall orientation of the proteins is such that proton pumping to the vesicle interior occurs upon illumination and after addition of ATP. All liposomes which contain H(+)-ATPase also contain bacteriorhodopsin. The rate of ATP hydrolysis was measured as function of pH in the dark and during illumination of the proteoliposomes. The pH dependency can be described by the protonation of a monovalent group from the outside with an apparent pK of 7.3 and the deprotonation of a monovalent group at the inside with an apparent pK of 3.7. Inside and outside refer to the orientation of the H(+)-ATPase in the liposomes which is opposite to that occurring in vivo. It is concluded that the first step in the reaction cycle is the binding of a proton from the cytosol which is followed by ATP binding, ATP hydrolysis on the enzyme and the release of ADP and phosphate, and finally the proton is released from the enzyme into the external medium.
Adenosine Diphosphate↗
Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.
Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.
Asparagine↗
In vitro synthesis of bacterio-opsin: integration into microsomal membranes.
The translation and membrane integration of bacterio-opsin from Halobacterium salinarium were investigated. Plasmids containing the bacterio-opsin-gene with or without its original presequence were transcribed with the T7-RNA-polymerase and translated in vitro in a wheat germ system. The integration of the expressed bacterio-opsin into dog pancreas microsomes was studied. Both precursor bacterio-opsin and mature bacterio-opsin integrate into the eukaryotic membrane.
Animals↗
Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.
Light-induced H+ release and reuptake as well as surface potential changes inherent in the bacterio-rhodopsin reaction cycle were measured between 10 degrees C and 50 degrees C. Signals of optical pH indicators covalently bound to Lys-129 at the extracellular surface of bacteriorhodopsin were compared with absorbance changes of probes residing in the aqueous bulk phase. Only surface-bound indicators monitor the kinetics of H+ ejection from bacteriorhodopsin and allow the correlation of the photocycle with the pumping cycle. During the L550----M412 transition the H+ appears at the extracellular surface of bacteriorhodopsin. Surface potential changes detected by bound fluorescein or by the potentiometric probe 4-[2-(di-n-butylamino)-6-naphthyl]vinyl-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS) occur in milliseconds concomitantly with the formation and decay of the N intermediate. pH indicators residing in the aqueous bulk phase reflect the transfer of H+ from the membrane surface into the bulk but do not probe the early events of H+ pumping. The observed retardation of H+ at the membrane surface for several hundred microseconds is of relevance for energy conversion of biological membranes powered by electrochemical H+ gradients.
Bacteriorhodopsins↗
Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.
The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X-ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH-values. The observed light-induced intensity changes of in-plane X-ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X-ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3-3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at -180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.
Bacteriorhodopsins↗
Bacteriorhodopsin in ice. Accelerated proton transfer from the purple membrane surface.
The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H(+)-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure.
Bacteriorhodopsins↗
Light- and dark-adapted bacteriorhodopsin, a time-resolved neutron diffraction study.
Recently, neutron diffraction experiments have revealed well-resolved and reversible changes in the protein conformation of bacteriorhodopsin (BR) between the light-adapted ground state and the M-intermediate of the proton pumping photocycle (Dencher, Dresselhaus, Zaccai and Büldt (1989) Proc. Natl. Acad. Sci. USA 86, 7876-7879). These changes are triggered by the light-induced isomerization of the chromophore retinal from the all-trans to the 13-cis configuration. Dark-adapted purple membranes contain a mixture of two pigment species with either the all-trans- or 13-cis-retinal isomer as chromophore. Employing a time-resolved neutron diffraction technique, no changes in protein conformation in the resolution regime of up to 7 A are observed during the transition between the two ground-state species 13-cis-BR and all-trans-BR. This is in line with the fact that the conversion of all-trans BR to 13-cis-BR involves an additional isomerization about the C15 = N Schiff's base bond, which in contrast to M formation minimizes retinal displacement and keeps the Schiff's base in the original protein environment. Furthermore, there is no indication for large-scale redistribution of water molecules in the purple membrane during light-dark adaptation.
Bacteriorhodopsins↗
Water molecules and exchangeable hydrogen ions at the active centre of bacteriorhodopsin localized by neutron diffraction. Elements of the proton pathway?
Neutron diffraction is used to localize water molecules and/or exchangeable hydrogen ions in the purple membrane by H2O/2H2O exchange experiments at different values of relative humidity. At 100% relative humidity, differences in the hydration between protein and lipid areas are observed, accounting for an excess amount of about 100 molecules of water in the lipid domains per unit cell. A pronounced isotope effect was observed, reproducibly showing an increase in the lamellar spacing from 60 A in 2H2O to 68 A in H2O. At 15% relative humidity, the positions of exchangeable protons became visible. A dominant difference density peak corresponding to 11 +/- 2 exchangeable protons was detected in the central part of the projected structure of bacteriorhodopsin at the Schiff's base end of the chromophore. A difference density map obtained from data on purple membrane films at 15% relative humidity in 2H2O, and the same sample after complete drying in vacuum, revealed that about eight of these protons belong to four water molecules. This is direct evidence for tightly bound water molecules close to the chromophore binding site of bacteriorhodopsin, which could participate in the active steps of H+ translocation as well as in the proton pathway across this membrane protein.
Bacteriorhodopsins↗