Protonation reactions and their coupling in bacteriorhodopsin.
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Light-induced changes of the proton affinities of amino acid side groups are the driving force for proton translocation in bacteriorhodopsin. Recent progress in obtaining structures of bacteriorhodopsin and its intermediates with an increasingly higher resolution, together with functional studies utilizing mutant pigments and spectroscopic methods, have provided important information on the molecular architecture of the proton transfer pathways and the key groups involved in proton transport. In the present paper I consider mechanisms of light-induced proton release and uptake and intramolecular proton transport and mechanisms of modulation of proton affinities of key groups in the framework of these data. Special attention is given to some important aspects that have surfaced recently. These are the coupling of protonation states of groups involved in proton transport, the complex titration of the counterion to the Schiff base and its origin, the role of the transient protonation of buried groups in catalysis of the chromophore's thermal isomerization, and the relationship between proton affinities of the groups and the pH dependencies of the rate constants of the photocycle and proton transfer reactions. (+info)
Chemical and physical evidence for multiple functional steps comprising the M state of the bacteriorhodopsin photocycle.
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In the photocycle of bacteriorhodopsin (bR), light-induced transfer of a proton from the Schiff base to an acceptor group located in the extracellular half of the protein, followed by reprotonation from the cytoplasmic side, are key steps in vectorial proton pumping. Between the deprotonation and reprotonation events, bR is in the M state. Diverse experiments undertaken to characterize the M state support a model in which the M state is not a static entity, but rather a progression of two or more functional substates. Structural changes occurring in the M state and in the entire photocycle of wild-type bR can be understood in the context of a model which reconciles the chloride ion-pumping phenotype of mutants D85S and D85T with the fact that bR creates a transmembrane proton-motive force. (+info)
Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle.
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Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP. Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics. Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution. We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date. A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics. (+info)
Structures of photointermediates and their implications for the proton pump mechanism.
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It is widely accepted that bacteriorhodopsin undergoes global conformational changes during its photocycle. In this review, the structural properties of the M and N intermediates are described in detail. Based on the clarified global conformational change, we propose a model for the molecular mechanism of the proton pump. The global structural change is suggested to be a key component in establishing vectorial proton transport. (+info)
Role of internal water molecules in bacteriorhodopsin.
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Internal water molecules are considered to play a crucial role in the functional processes of proton pump proteins. They may participate in hydrogen-bonding networks inside proteins that constitute proton pathways. In addition, they could participate in the switch reaction by mediating an essential proton transfer at the active site. Nevertheless, little has been known about the structure and function of internal water molecules in such proteins. Recent progress in infrared spectroscopy and X-ray crystallography provided new information on water molecules inside bacteriorhodopsin, the light-driven proton pump. The accumulated knowledge on bacteriorhodopsin in the last decade of the 20th century will lead to a realistic picture of internal water molecules at work in the 21st century. In this review, I describe how the role of water molecules has been studied in bacteriorhodopsin, and what should be known about the role of water molecules in the future. (+info)
Water and bacteriorhodopsin: structure, dynamics, and function.
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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. (+info)
Analogies between halorhodopsin and bacteriorhodopsin.
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The light-activated proton-pumping bacteriorhodopsin and chloride ion-pumping halorhodopsin are compared. They belong to the family of retinal proteins, with 25% amino acid sequence homology. Both proteins have seven alpha helices across the membrane, surrounding the retinal binding pocket. Photoexcitation of all-trans retinal leads to ion transporting photocycles, which exhibit great similarities in the two proteins, despite the differences in the ion transported. The spectra of the K, L, N and O intermediates, calculated using time-resolved spectroscopic measurements, are very similar in both proteins. The absorption kinetic measurements reveal that the chloride ion transporting photocycle of halorhodopsin does not have intermediate M characteristic for deprotonated Schiff base, and intermediate L dominates the process. Energetically the photocycle of bacteriorhodopsin is driven mostly by the decrease of the entropic energy, while the photocycle of halorhodopsin is enthalpy-driven. The ion transporting steps were characterized by the electrogenicity of the intermediates, calculated from the photoinduced transient electric signal measurements. The function of both proteins could be described with the 'local access' model developed for bacteriorhodopsin. In the framework of this model it is easy to understand how bacteriorhodopsin can be converted into a chloride pump, and halorhodopsin into a proton pump, by changing the ion specificity with added ions or site-directed mutagenesis. (+info)
Proton transport by sensory rhodopsins and its modulation by transducer-binding.
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The study of light-induced proton transfers in the archaeal sensory rhodopsins (SR), phototaxis receptors in Halobacterium salinarum, has contributed important insights into their mechanism of signaling to their cognate transducer subunits in the signaling complex. Essential features of the bacteriorhodopsin (BR) pumping mechanism have been conserved in the evolution of the sensors, which carry out light-driven electrogenic proton transport when their transducers are removed. The interaction of SRI with its transducer blocks proton-conducting channels in the receptor thereby inhibiting its proton pumping, indicating that the pump machinery, rather than the transport activity itself, is functionally important for signaling. Analysis of SRII mutants has shown that the salt bridge between the protonated Schiff base and its counterion Asp73 constrains the receptor in its inactive conformation. Similarly, in BR, the corresponding salt bridge between the protonated Schiff base and Asp85 contributes to constraining the protein in a conformation in which its cytoplasmic channel is closed. Transducer chimera studies further indicate that the receptor conformational changes are transmitted from the sensors to their cognate transducers through transmembrane helix-helix interaction. These and other results reviewed here support a signaling mechanism in which tilting of helices on the cytoplasmic side (primarily outward tilting of helix F), similar to that which occurs in BR in its open cytoplasmic channel conformation, causes structural alterations in the transducer transmembrane helices. (+info)