ABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/metabolism , Cation Transport Proteins/metabolism , Escherichia coli Proteins/metabolism , Potassium/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/metabolism , Cation Transport Proteins/chemistry , Detergents/chemistry , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Ion Transport , Kinetics , Liposomes/chemistry , Liposomes/metabolism , Models, Molecular , Phosphorylation , Protein Binding , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , Protons , Pyridinium Compounds/chemistry , Sodium/metabolism , Styrenes/chemistry , ThermodynamicsABSTRACT
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-ß-D-maltoside ('C12-b-M') as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. 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.
