The key function of energy-transducing membrane proteins is the creation of a proton gradient by directional proton transfer. The role of protein-bound water molecules herein is not fully understood, as X-ray diffraction analysis has resolved the positions of oxygen, but not of hydrogen atoms in such protein-water complexes. Here we show, now timeresolved at atomic resolution, how a membrane protein achieves directional proton transfer via protein-bound water molecules in contrast to random proton transfer in liquid water. A combination of X-ray structure analysis, timeresolved FTIR spectroscopy, and molecular dynamics (MD) simulations elucidates how directionality is achieved. Using the proton-pump bacteriorhodopsin as the paradigm, we show how controlled conformational changes of few amino acid residues rearrange preordered water molecules and induce directional proton transfer. This mechanism is analogous to an electronic diode: a "proton diode".According to the chemiosmotic theory, the creation of a proton gradient in photosynthesis [1] and oxidative phosphorylation [2][3][4] by means of directional proton transfer is the key step for energy transduction in living cells.

The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K(+) and H(+) were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K(+) concentrations and pK values for H(+) binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K(+) into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post-Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.

Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties.Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside ("C12-b-M") as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors by approximately 10°C compared to C12-b-M. Another surfactant yielded a stabilization of the human Patched protein receptor by 13°C.In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b 6 f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. Declaration of Interest statementThe authors report no declarations of interest. Mixed protein-surfactant micelles are often used for in vitro studies when solutions of pure, monodisperse integral membrane proteins are needed (Garavito and Ferguson-Miller, 2001). Because integral membrane proteins evolved in a lipid bilayer environment, the exchange of lipid for surfactant frequently causes a destabilization of the protein. This can result in increased activity followed by increased rate of denaturation, increased susceptibility to protease attack, accelerated heat denaturation, oxidative damage, loss of activity regulation and lack of crystallization (Tanford and Reynolds, 1976;Tate, 2010). Europe PMC Funders GroupAt the beginning of membrane protein research only surfactants developed as detergents for consumer or industrial use were available many of which were inhomogenous. In-vitro studies of solubilized membrane proteins and structural work (Rosenbusch et al., 1981;Michel and Oesterhelt, 1980) led to an increasing demand in tailored surfactants. In the micellar solution the protein should be functional and stable for extended periods of time and the micellar belt attached to the protein should be small in order to leave much of the polar protein surfaces uncovered for crystal lattice contacts (Michel, 1983).Studies of the micellar mass revealed the relation between the chemical structure of a surfactant and the size of its micelle. It was found to depend upon the ratio of the polar cross-section and the length of the extended hydrophobic chain (Israelachvili et al., 1976). In case of spherical micelles the latter matches the radius of the hydrophobic core. With alkyl chains as hydrophobic moieties of surfactants proteins require a length between an extended octyl and a dodecyl chain (Iwata, 2003).Small micelles are for...

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