RESUMEN
By the elucidation of high-resolution structures the view of the bioenergetic processes has become more precise. But in the face of these fundamental advances, many problems are still unresolved. We have examined a variety of aspects of energy-transducing membranes from large protein complexes down to the level of protons and functional relevant picosecond protein dynamics. Based on the central role of the ATP synthase for supplying the biological fuel ATP, one main emphasis was put on this protein complex from both chloroplast and mitochondria. In particular the stoichiometry of protons required for the synthesis of one ATP molecule and the supramolecular organisation of ATP synthases were examined. Since formation of supercomplexes also concerns other complexes of the respiratory chain, our work was directed to unravel this kind of organisation, e.g. of the OXPHOS supercomplex I(1)III(2)IV(1), in terms of structure and function. Not only the large protein complexes or supercomplexes work as key players for biological energy conversion, but also small components as quinones which facilitate the transfer of electrons and protons. Therefore, their location in the membrane profile was determined by neutron diffraction. Physico-chemical features of the path of protons from the generators of the electrochemical gradient to the ATP synthase, as well as of their interaction with the membrane surface, could be elucidated by time-resolved absorption spectroscopy in combination with optical pH indicators. Diseases such as Alzheimer's dementia (AD) are triggered by perturbation of membranes and bioenergetics as demonstrated by our neutron scattering studies.
Asunto(s)
Adenosina Trifosfato/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Metabolismo Energético , Membranas Mitocondriales/metabolismo , ATPasas de Translocación de Protón de Cloroplastos/química , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Humanos , Luz , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosforilación Oxidativa , Protones , Escualeno/análogos & derivados , Escualeno/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
Membrane integral ATP synthases produce adenosine triphosphate, the universal "energy currency" of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it.
Asunto(s)
ATPasas de Translocación de Protón Mitocondriales/ultraestructura , Proteínas de Plantas/ultraestructura , Estructura Cuaternaria de Proteína , Adenosina Trifosfato/biosíntesis , Cloroplastos/enzimología , Coenzimas/metabolismo , Cristalografía por Rayos X , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Conformación Proteica , Subunidades de Proteína/metabolismo , Spinacia oleracea/enzimología , Ubiquinona/metabolismoRESUMEN
The fluorescence quenching of 9-aminoacridine (9-AA) after imposition of a transmembrane pH gradient (inside acidic) in liposomes has been investigated for a number of different lipid systems. The initial fluorescence decrease after a rapid pH jump, induced in the extravesicular medium by a stopped-flow mixing technique, was ascribed to a response of 9-AA to the imposed pH gradient and not to changes in the vesicular system itself. Time constants for this fluorescence quenching are in the range of several hundred milliseconds at 25 degrees C. Fluorescence recovery which should be correlated to the dissipation of the pH gradient occurs in the 100 s time range and is 10-30-times faster than the delta pH decay monitored with the entrapped hydrophilic pH-indicator dye pyranine. The quenching was severely hindered below the lipid phase transition of dipalmitoylphosphatidylglycerol. No delta pH-induced quenching was obtained in lipid vesicles containing only zwitterionic, net uncharged phosphatidylcholine headgroups. For the occurrence of quenching, the presence of negatively charged headgroups, i.e. phosphatidylglycerol or phosphatidylserine, was necessary. The extent of quenching, at a specific pH difference applied, had a cooperative dependency (Hill coefficient approximately 2) on the number of negative headgroups in the membrane and on the concentration of unquenched (unbound) 9-AA molecules. The concentration of quenched 9-AA molecules was furthermore proportional to the number of dimer-excimer complexes of 9-AA which are formed during the quenching process.
Asunto(s)
Aminacrina/metabolismo , Aminoacridinas/metabolismo , Liposomas/metabolismo , 1,2-Dipalmitoilfosfatidilcolina , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Dimiristoilfosfatidilcolina , Fluorescencia , Concentración de Iones de Hidrógeno , Cinética , Membrana Dobles de Lípidos/metabolismo , Sustancias Macromoleculares , Fosfatidilgliceroles , Fosfolípidos , Cloruro de Potasio/farmacología , Espectrometría de Fluorescencia , Temperatura , Termodinámica , Valinomicina/farmacologíaRESUMEN
A wealth of information has been gathered during the past decades that water molecules do play an important role in the structure, dynamics, and function of bacteriorhodopsin (bR) and purple membrane. Light-induced structural alterations in bR as detected by X-ray and neutron diffraction at low and high resolution are discussed in relationship to the mechanism of proton pumping. The analysis of high resolution intermediate structures revealed photon-induced rearrangements of water molecules and hydrogen bonds concomitant with conformational changes in the chromophore and the protein. These observations led to an understanding of key features of the pumping mechanism, especially the vectoriality and the different modes of proton translocation in the proton release and uptake domain of bR. In addition, water molecules influence the function of bR via equilibrium fluctuations, which must occur with adequate amplitude so that energy barriers between conformational states can be overcome.
Asunto(s)
Bacteriorodopsinas/química , Agua/química , Cristalografía , Modelos Químicos , Fotoquímica , Conformación Proteica , Bombas de Protones/química , Membrana Púrpura/química , TermodinámicaRESUMEN
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.
Asunto(s)
Bacteriorodopsinas , Adaptación a la Oscuridad , Luz , Fenómenos Químicos , Química Física , Halobacterium/análisis , Neutrones , Conformación Proteica , Análisis Espectral , TemperaturaRESUMEN
The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the alpha-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.
Asunto(s)
Aminoácidos , Bacteriorodopsinas/análisis , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Conformación Proteica , Difracción de Rayos XRESUMEN
The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.
Asunto(s)
Cloroplastos/enzimología , Indicadores y Reactivos , ATPasas de Translocación de Protón/química , Colorantes de Rosanilina , Spinacia oleracea/enzimología , Catálisis , Cristalización , Electroforesis en Gel de Poliacrilamida/métodos , Indicadores y Reactivos/química , Estructura Molecular , Estructura Secundaria de Proteína , ATPasas de Translocación de Protón/aislamiento & purificación , ATPasas de Translocación de Protón/metabolismo , Colorantes de Rosanilina/químicaRESUMEN
The proton-conducting pathway of bacteriorhodopsin (BR) contains at least nine internal water molecules that are thought to be key players in the proton translocation mechanism. Here, we report the results of a multinuclear (1H, 2H, 17O) magnetic relaxation dispersion (MRD) study with the primary goal of determining the rate of exchange of these internal water molecules with bulk water. This rate is of interest in current attempts to elucidate the molecular details of the proton translocation mechanism. The relevance of water exchange kinetics is underscored by recent crystallographic findings of substantial variations in the number and locations of internal water molecules during the photocycle. Moreover, internal water exchange is believed to be governed by conformational fluctuations in the protein and can therefore provide information about the thermal accessibility of functionally important conformational substates. The present 2H and 17O MRD data show that at least seven water molecules, or more if they are orientationally disordered, in BR have residence times (inverse exchange rate constant) in the range 0.1-10 micros at 277 K. At least five of these water molecules have residence times in the more restrictive range 0.1-0.5 micros. These results show that most or all of the deeply buried water molecules in BR exchange on a time-scale that is short compared to the rate-limiting step in the photocycle. The MRD measurements were performed on BR solubilized in micelles of octyl glucoside. From the MRD data, the rotational correlation time of detergent-solubilized BR was determined to 35 ns at 300 K, consistent with a monomeric protein in complex with about 150 detergent molecules. The solubilized protein was found to be stable in the dark for at least eight months at 277 K.
Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Halobacterium salinarum/química , Agua/metabolismo , Detergentes/metabolismo , Deuterio/metabolismo , Enlace de Hidrógeno , Cinética , Micelas , Modelos Moleculares , Conformación Proteica , Rotación , Solubilidad , Agua/químicaRESUMEN
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.
Asunto(s)
Bacteriorodopsinas , Sitios de Unión , Transporte Biológico , Cristalografía/métodos , Hidrógeno , Neutrones , Protones , AguaRESUMEN
Neutron diffraction from oriented purple membrane fragments at various hydration levels, coupled with H2O/2H2O exchange, was used to compare the structure and hydration of the light-adapted initial state (B-state) and the M photointermediate of bacteriorhodopsin mutant D96N. Diffraction patterns were recorded at 86%, 75% and 57% relative humidity (r.h.). Structural changes observed at 86% and 75% r.h. are absent at 57% r.h., showing that they are uncoupled from the deprotonation of the Schiff base during formation of the M-state. In a current model, the M-state consists of two substates, M1 and M2. Our data suggest that the state trapped at 57% r.h. is M1 and that M2 is trapped at the higher r.h. values. The observed structural changes are, therefore, associated with the M1-->M2 transition, which can only take place at higher r.h. The difference Fourier projections of exchangeable hydrogen atoms and water molecules in the membrane plane are very similar for the B and M-states at 75% and 86% r.h. This shows that contrary to certain models, the structural changes in the M-state are not correlated with major hydration changes in the proton channel projection.
Asunto(s)
Bacteriorodopsinas/genética , Bacteriorodopsinas/metabolismo , Agua/metabolismo , Asparagina/genética , Ácido Aspártico/genética , Análisis de Fourier , Halobacterium salinarum , Mutación , Neutrones , Membrana Púrpura/química , Membrana Púrpura/metabolismo , Bases de Schiff/química , Difracción de Rayos XRESUMEN
The lamellar spacing dl of purple membrane (PM) multilayer systems was investigated with neutron diffraction as a function of temperature and of the level of hydration. The observed large T-dependent variations of dl indicate that PM is partially dehydrated when cooled below a "hydration water freezing point". This phenomenon is reversible, but a hysteresis is observed when PM is rehydrated upon reheating. The hydration water remaining bound to the membrane below about 240 K is non-freezing. Its amount was found to be hnf=0.24(+/-0.02) g 2H2O/g BR for all samples equilibrated at room temperature in the presence of 2H2O vapour at >/=84% r.h. It is evident, that the dehydration/rehydration behaviour of PM is strongly correlated with the temperature-dependent behaviour of the dynamical structure factor. Above the well-known "dynamical transition" announcing the onset of localized diffusive molecular motions between 190 K and 230 K, a second dynamical transition is caused by the temperature-induced rehydration of the PM starting near 255 K. This is also correlated with the deviation from a pure Arrhenius law of the rate-limiting process in the photocycle, known to occur upon cooling beyond the ice point into the same temperature region. Our results suggest that the phenomenon of dehydration and rehydration induced by cooling and reheating, respectively, is a general property of biological membranes.
Asunto(s)
Membrana Celular/química , Congelación , Halobacterium salinarum/química , Desecación , Halobacterium salinarum/fisiología , Membrana Dobles de LípidosRESUMEN
Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.
Asunto(s)
Halobacterium salinarum/citología , Hidroxilamina/metabolismo , Microscopía de Fuerza Atómica , Membrana Púrpura/metabolismo , Membrana Púrpura/ultraestructura , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Bacteriorodopsinas/ultraestructura , Cristalización , Halobacterium salinarum/ultraestructura , Hidroxilamina/farmacología , Procesamiento de Imagen Asistido por Computador , Unión Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Membrana Púrpura/química , Membrana Púrpura/efectos de los fármacosRESUMEN
X-ray diffraction patterns have been recorded from a single layer of purple membrane ( approximately 50 A thickness) at the air/water interface in a Langmuir trough. Grazing-incidence X-ray diffraction is demonstrated to be a promising method for obtaining structural information on membrane proteins under physiological conditions. The method is so sensitive that diffraction can be measured from samples with only 10(13) protein molecules in the beam. Diffraction from hexagonal crystals of purple membrane with a lattice constant of 61. 3 A was observed up to the order {h,k}={4,3}, corresponding to a resolution of approximately 9 A. The work reported here is a first step towards a new way of protein crystallography using grazing-incidence X-ray diffraction at the air/water interface.
Asunto(s)
Membrana Púrpura/química , Difracción de Rayos X/métodos , Aire , Bacteriorodopsinas/química , Cristalografía/métodos , Halobacterium salinarum/ultraestructura , Microscopía Fluorescente , Membrana Púrpura/ultraestructura , Propiedades de Superficie , Agua , Difracción de Rayos X/instrumentaciónRESUMEN
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.
Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/fisiología , Halobacterium/fisiología , Transporte Biológico Activo , Difusión , Concentración de Iones de Hidrógeno , Hielo , Cinética , LuzRESUMEN
By neutron scattering experiments and time-resolved absorption spectroscopy we have investigated picosecond equilibrium fluctuations and the kinetics of the photocycle of bacteriorhodopsin (BR) in the purple membrane (PM). Natural PM samples composed of 75% BR (w/w) and 25% lipid (w/w) as well as delipidated PM having only 5% lipid (w/w) were measured at different levels of hydration. We observed a reduced 'flexibility', due to a diminished weight of stochastic large-amplitude motions occurring in the delipidated PM as compared to the natural PM. This effect is more pronounced for wet samples, indicating the importance of lipid hydration for protein dynamics. The reduced flexibility is accompanied by significantly larger time constants describing the decay of the M-intermediate. Therefore, a correlation between the dynamical behavior of the protein-lipid complex and BR function emerges.
Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Transferencia de Energía , Halobacterium salinarum/química , Cinética , Lípidos , Modelos Químicos , Neutrones , Dispersión de Radiación , Factores de Tiempo , AguaRESUMEN
Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.
Asunto(s)
Membrana Celular/química , Complejos Multienzimáticos/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Complejos de ATP Sintetasa , Cloroplastos/química , Electroquímica , Electroforesis en Gel de Poliacrilamida , Microscopía de Fuerza Atómica , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Tinción con Nitrato de Plata , Spinacia oleracea/enzimologíaRESUMEN
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.