Extraction and liposome reconstitution of membrane proteins with their native lipids without the use of detergents.

Functional studies of membrane-bound channels, transporters or signal transducers require that the protein of interest resides in a membrane that separates two compartments. One approach that is commonly used to prepare these systems is to reconstitute the protein in liposomes. An intermediate step of this method is purification of the protein, which typically involves solubilization of the native membrane using detergent. The use of detergents often results in removal of lipids surrounding the protein, which may alter its structure and function. Here, we have employed a method for isolation of membrane proteins with a disc of their native lipids to develop an approach that allows transfer of the purified membrane protein to liposomes without the use of any detergents.

Isolation of yeast complex IV in native lipid nanodiscs.

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11nm×14nm. Based on this size we estimated that each CytcO was surrounded by ~100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc1 in the mitochondrial membrane, cyt. bc1 was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O2-reduction activity of ~130 electrons CytcO-1s-1 and the kinetics of the reaction of the fully reduced CytcO with O2 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

Electron and proton transfer in the ba(3) oxidase from Thermus thermophilus.

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O(2) to water is catalysed at a haem a(3)-Cu(B) catalytic site. The three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa(3) oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba( 3 )-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k congruent with 6.8 x 10(4) s(-1)) and formation of the peroxy (P(R)) intermediate. In the next step a proton was taken up from solution with a rate constant of approximately 1.7 x 10(4) s(-1), associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of approximately 1,100 s(-1), accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was approximately 1.5 protons per enzyme molecule. The results support the earlier proposal that the P(R) and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba(3) oxidases, the proton-uptake reactions occur over the same time scales as in the aa(3)-type oxidases.

Development of a bacterial biosensor for nitrotoluenes: the crystal structure of the transcriptional regulator DntR.

The transcriptional regulator DntR, a member of the LysR family, is a central element in a prototype bacterial cell-based biosensor for the detection of hazardous contamination of soil and groundwater by dinitrotoluenes. To optimise the sensitivity of the biosensor for such compounds we have chosen a rational design of the inducer-binding cavity based on knowledge of the three-dimensional structure of DntR. We report two crystal structures of DntR with acetate (resolution 2.6 angstroms) and thiocyanate (resolution 2.3 angstroms), respectively, occupying the inducer-binding cavity. These structures allow for the construction of models of DntR in complex with salicylate (Kd approximately or = 4 microM) and 2,4-dinitrotoluene that provide a basis for the design of mutant DntR with enhanced specificity for dinitrotoluenes. In both crystal structures DntR crystallises as a homodimer with a "head-to-tail" arrangement of monomers in the asymmetric unit. Analysis of the crystal structure has allowed the building of a full-length model of DntR in its biologically active homotetrameric form consisting of two "head-to-head" dimers. The implications of this model for the mechanism of transcription regulation by LysR proteins are discussed.