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We present an approach that allows rapid determination of the topology of Escherichia coli inner-membrane proteins by a combination of topology prediction and limited fusion-protein analysis. We derive new topology models for 12 inner-membrane proteins: MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA. We estimate that our approach should make it possible to arrive at highly reliable topology models for roughly 10% of the Ϸ800 inner-membrane proteins thought to exist in E. coli.bioinformatics ͉ fusion protein A n important first step in the characterization of an integral membrane protein of the helix bundle class (1) is to determine its membrane topology-i.e., the number of transmembrane ␣-helices and the overall in͞out orientation of the protein relative to the membrane. In Escherichia coli, this step is usually accomplished by using reporter enzymes such as PhoA or LacZ fused to different portions of the membrane protein (2). In general, the number of fusions that need to be made and analyzed for a complete topology determination is equal to or larger than the number of transmembrane helices in the protein, thus requiring a significant experimental effort.In the absence of experimental information, one can use various topology prediction methods to gain an idea of a protein's topology. The best current methods predict the correct topology with a success rate of 65-70% (3, 4) and thus provide a reasonable guide to minimizing the number of fusion proteins that have to be made for a given membrane protein (5). Recently, we have shown that the reliability of a given topology prediction can be estimated by comparing the predictions from a number of different prediction programs (6): when all methods agree, the topology is virtually certain to be correct, whereas the fraction of correct predictions drops with increasing levels of disagreement between the different methods.Here, we suggest that the amount of experimental work needed to establish a topology should be inversely related to the reliability of the theoretical topology prediction, and we provide data that allows the topology for 12 E. coli inner-membrane proteins to be deduced from a combination of topology predictions and single C-terminal reporter-protein fusions. Given that there are only Ϸ60 experimentally determined topologies for E. coli inner-membrane proteins available in the literature (6, 7), our 12 additional topologies represent a substantial increase in topology information. From topology predictions for the whole complement of E. coli inner-membrane proteins (Ϸ800 proteins), we estimate that the topology for an additional Ϸ75 proteins can be rapidly mapped by using our approach. (10) DNA Techniques. All plasmid constructs were confirmed by DNA sequencing using T7 DNA polymerase. The genes encoding the E. coli MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA proteins were amplified from E. coli JM109 by using Taq polymerase. The genes were cloned by using primer-introduced sites 5Ј XhoI and...

Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

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 11 nm x 14 nm. 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 bc 1 in the mitochondrial membrane, cyt. bc 1 was not found in the native nanodiscs. Yet, the loosely-bound Respiratory