The role of specific lipid structures in biological membranes has been elusive. There are hundreds of them in nature. Why has nature made them? How do they aid in the functioning of membrane proteins? Genetics with its 'knock out' organisms declares that functions persist in the absence of any particular lipid. Nonetheless some lipids, such as cardiolipin (CL), are associated with particular functions in the cell. It may merely expand the variety of culture conditions (pH, temperature, etc.) under which the wild-type organism survives. This article explores a unique role of CL as a proton trap within membranes that conduct oxidative phosphorylation and therefore the synthesis of ATP. CL's pK(2) (above 8.0) provides a role for it as a headgroup proton trap for oxidative phosphorylation. It suggests why CL is found in membranes that pump protons. The high pK(2) also indicates that the headgroup has but one negative charge in the neutral pH range. Data on the binding of CL to all of the oxidative phosphorylation proteins suggest that the CL may aggregate the oxidative phosphorylation proteins into a patch while it restricts pumped protons within its headgroup domain - supplying protons to the ATP synthase with minimal changes in the bulk phase pH.

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.

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To elucidate general features of structural and dynamical properties of hydration water and the influence of hydration water on the dynamical behavior of biomembranes, purple membranes from halobacteria and disk membranes from bovine retinae have been studied by neutron scattering techniques. Hydrated films of oriented multilamellar membrane stacks were used to measure lamellar diffraction patterns and quasielastic incoherent neutron scattering as a function of hydration level, of temperature, and of the protein/lipid ratio. These measurements revealed a strong interaction of a "first hydration layer" with the membrane surface and a reduced self-diffusion of aqueous solvent parallel to the membrane surface (the self-diffusion coefficient is about 5 times smaller as compared to excess water). The picosecond internal molecular motions of the protein/ lipid complex are strongly affected by the amount of solvent interacting with the lipids and the membrane proteins. In particular, the lipids and their ability to attract solvent molecules play an important role for "hydration-induced flexibility" of biomembranes. On the basis of these measurements, the impact of the hydration process on the function of biomembranes is discussed for the light-driven proton pump bacteriorhodopsin in purple membranes.

A neutron diffraction study of spectroscopic states for the light-energized proton pump bacteriorhodopsin (BR) is presented. The photocycle states BR-568 and M were generated at temperatures above 40C and were measured after trapping at -1800C. In the BR-568 to M-state transition, which is known to be a key step in transmembrane proton pumping, reversible structural changes of the protein were detected. These structural alterations occur in the neighborhood of the cyclohexene ring and at the Schiff's base end of the chromophore retinal. They are interpreted as a 1-2°tilt of three or four of the transmembrane a-helices or as positional changes of four or five amino acids. The structural changes observed are inherent in the transport mechanism of bacteriorhodopsin.Membrane protein conformational changes are expected to be involved in transport mechanisms. For the light-driven proton pump bacteriorhodopsin (BR), however, the all-trans to 13-cis isomerization of the chromophore retinal, during the first steps of the photocycle, is the only structural alteration unambiguously determined to date (1, 2). A variety of spectroscopic and diffraction experiments have been performed to detect changes also in the protein moiety of BR connected with the transition from the BR-568 ground state to the photocycle intermediate M. These efforts resulted in contradictory ideas about the extent of the conformational changes occurring. They -range from lattice disorder and dramatic changes in the tertiary conformation to only subtle alterations in the structure at high resolution (for review, see ref.3). To settle this controversy, we have conducted a neutron diffraction study on different functional states of BR. The advantages of neutron diffraction for the study of biological membranes derive mainly from the natural contrast between protein and protein-associated water as well as between protein and lipid, which is higher for neutrons than for x-rays. Furthermore, since the coherent neutron scattering length of H (-3.74 fm) and 2H (6.67 fm) is different not only in magnitude but also in sign, there are rich possibilities of 2H labeling. By performing measurements in H20 and 2H20 on the BR-568 ground state and the M intermediate, it should be possible not only to detect structural differences in the protein but also to observe any redistribution of associated water and exchangeable hydrogens related to the transition.The results obtained by neutron diffraction unambiguously show that in the BR-568 to M transition, which is known to be a key step in transmembrane proton pumping, reversible structural changes of the protein in the vicinity of the chromophore retinal occur. These light-induced conformational changes are proposed to trigger the vectorial H+ translocation processes in the active center of BR. METHODSSample Preparation. Purple membranes were isolated from Halobacterium halobium (strain ET 1001). Neutron diffraction experiments require '100 mg of BR to perform the experiments in a reasonable time of ""2 days for t...

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I 1 III 2 and I 1 III 2 IV 1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I 1 III 2 and I 1 III 2 IV 1 closely fits the x-ray structure of monomeric complex IV and shows its location in the assembly. By comparing different views of supercomplex I 1 III 2 IV 1 , the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.All living organisms use a series of integral membrane protein complexes for energy conversion and ATP synthesis. In eukaryotes, electrons are transported by the respiratory chain, starting from NADH via complex I (NADH:ubiquinone oxidoreductase) or from succinate via complex II (succinate:ubiquinone oxidoreductase), the membrane integral electron carrier ubiquinol, complex III (ubiquinol:cytochrome c oxidoreductase), the peripheral electron carrier cytochrome c, and complex IV (cytochrome c oxidase) to the terminal acceptor molecular oxygen (1). The electron transport chain generates a proton gradient across the inner mitochondrial membrane, which is used by complex V (F O F 1 -ATP synthase) to synthesize ATP. In the last decade, structures of the individual respiratory chain complexes from various organisms have been determined. Atomic models exist for bovine heart mitochondrial complex III (2) and IV (3). A high resolution structure of complex I is not yet available, but electron microscopy indicates that it is L-shaped in all organisms investigated, and a 2.2-nm resolution map from cryoelectron microscopy exists for the bovine heart complex I (4).Two alternative models for the arrangement of the respiratory chain complexes in the membrane have been proposed. According to the currently favored random collision model (5), all components of the respiratory chain diffuse individually in the membrane, and electron transfer depends on the random, transient encounter of the individual protein complexes and the smaller electron carriers. In the solid state model (6) proposed 50 years ago, the substrate is channeled directly from one enzyme to the next. Recently isolated stoichiometric assemblies, so-called supercomplexes, support this model. Respiratory supercomplexes of different compositions have been described in bact...

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.

Matrix-assisted laser desorption/ionization (MALDI) mass spectra and methods to improve their quality are reported for three hydrophobic, membrane-bound proteins: porin from Escherichiu coli, bacteriorhodopsin from Halobacterium salinarium and cholesterolesterase from Pseudomonas Juorescens. Several commonly used UV and IR matrices have been tested. In addition, the susceptibility of MALDI mass spectrometry to various neutral and ionic detergents, known usually to degrade the quality of MALDI mass spectra, has been tested systematically. For porin, consisting of three identical noncovalently bound subunits, a new sample preparation is reported, resulting in the desorption of the intact quaternary protein structure. This leads to a better understanding of the way a given analyte is embedded into the host matrix crystals.