Use of Helical Wheels to Represent the Structures of Proteins and to Identify Segments with Helical Potential

The three-dimensional structures of α-helices can be represented by two-dimensional projections which we call helical wheels. Initially, the wheels were employed as graphical restatements of the known structures determined by Kendrew, Perutz, Watson, and their colleagues at the University of Cambridge and by Phillips and his coworkers at The Royal Institution. The characteristics of the helices, discussed by Perutz et al. (1965), and Blake et al. (1965), can be readily visualized by examination of these wheels. For example, the projections for most helical segments of myoglobin, hemoglobin, and lysozyme have distinctive hydrophobic arcs. Moreover, the hydrophobic residues tend to be clustered in the n ± 3, n, n ± 4 positions of adjacent helical turns. Such hydrophobic arcs are not observed when the sequences of nonhelical segments are plotted on the wheels. Since the features of these projections are also distinctive, however, the wheels can be used to divide sequences into segments with either helical or nonhelical potential. The sequences of insulin, cytochrome c, ribonuclease A, chymotrypsinogen A, tobacco mosaic virus protein, and human growth hormone were chosen for application of the wheels for this purpose

A novel <it>Geobacteraceae</it>-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe (III) and Mn (IV) oxides in <it>Geobacter sulfurreducens</it>

<p>Abstract</p> <p>Background</p> <p>Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism <it>Geobacter sulfurreducens </it>are of interest to understand the mechanisms of Fe(III) reduction in the <it>Geobacter </it>species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of <it>G. sulfurreducens</it>, OmpJ, is not a cytochrome yet it is required for metal respiration.</p> <p>Results</p> <p>When outer membrane proteins of <it>G. sulfurreducens </it>were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other <it>Geobacteraceae</it>, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the <it>ompJ </it>gene in <it>G. sulfurreducens </it>produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III) and insoluble Mn (IV) oxide, as the electron acceptor. The heme <it>c </it>content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing <it>c</it>-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal reduction.</p> <p>Conclusion</p> <p>OmpJ is a putative porin found in the outer membrane of the model metal reducer <it>G. sulfurreducens </it>that is required for respiration of extracellular electron acceptors such as soluble and insoluble metals. The effect of OmpJ in extracellular electron transfer is indirect, as OmpJ is required to keep the integrity of the periplasmic space necessary for proper folding and functioning of periplasmic and outer membrane electron transport components. The exclusive presence of <it>ompJ </it>in members of the <it>Geobacteraceae </it>family as well as its role in metal reduction suggest that the <it>ompJ </it>sequence may be useful in tracking the growth or activity of <it>Geobacteraceae </it>in sedimentary environments.</p

Rational engineering of Geobacter sulfurreducens electron transfer components: a foundation for building improved Geobacter-based bioelectrochemical technologies

Multiheme cytochromes have been implicated in Geobacter sulfurreducens extracellular electron transfer (EET). These proteins are potential targets to improve EET and enhance bioremediation and electrical current production by G. sulfurreducens. However, the functional characterization of multiheme cytochromes is particularly complex due to the co-existence of several microstates in solution, connecting the fully reduced and fully oxidized states. Over the last decade, new strategies have been developed to characterize multiheme redox proteins functionally and structurally. These strategies were used to reveal the functional mechanism of G. sulfurreducens multiheme cytochromes and also to identify key residues in these proteins for EET. In previous studies, we set the foundations for enhancement of the EET abilities of G. sulfurreducens by characterizing a family of five triheme cytochromes (PpcA-E). These periplasmic cytochromes are implicated in electron transfer between the oxidative reactions of metabolism in the cytoplasm and the reduction of extracellular terminal electron acceptors at the cell's outer surface. The results obtained suggested that PpcA can couple e−/H+ transfer, a property that might contribute to the proton electrochemical gradient across the cytoplasmic membrane for metabolic energy production. The structural and functional properties of PpcA were characterized in detail and used for rational design of a family of 23 single site PpcA mutants. In this review, we summarize the functional characterization of the native and mutant proteins. Mutants that retain the mechanistic features of PpcA and adopt preferential e−/H+ transfer pathways at lower reduction potential values compared to the wild-type protein were selected for in vivo studies as the best candidates to increase the electron transfer rate of G. sulfurreducens. For the first time G. sulfurreducens strains have been manipulated by the introduction of mutant forms of essential proteins with the aim to develop and improve bioelectrochemical technologies.This work was supported by project grants: PTDC/BBB-BEP/0753/2012 (to CS), L'Oréal Portugal Medals of Honor for Women in Science 2012 (to LM), SFRH/BD/89701/2012 (to JD), UID/Multi/04378/2013 from Fundação para a Ciência e a Tecnologia (FCT), Portugal and CTQ2011-22514 from the Ministerio De Economia y Competitividad. Cytochrome work at Argonne National Laboratory (MS, YL, and PP) was previously supported by the DOE Office of Biological and Environmental Research program. Currently, PP is partially supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy under contract no. DE-AC02-06CH11357. Geobacter sulfurreducens cells and cloning protocols referred in this work were kindly provided by Prof. Derek Lovley from the University of Massachusetts Amherst (USA).Peer reviewedPeer Reviewe

Preliminary Crystallographic Data on the Human λIII Bence Jones Protein Dimer Cle

A complete human λ Bence Jones protein dimer (Cle) has been isolated and crystallized. Protein Cle was characterized immunochemically and chemically as having a variable region amino acid sequence associated with light chains of the λ chain subgroup, λIII, and a constant region sequence characteristic of non-Mcg type λ chains. Bence Jones protein Cle contains two covalently bound intact monomers, each having a molecular weight of ~23,000. Crystals of Bence Jones protein Cle, obtained from ammonium sulfate solutions, diffract to 2.6 Å resolution and have the orthorhombic space group P212121 with cell dimensions a = 113.0 A ̊, b = 72.3 A ̊, and c = 48.9 A ̊. The asymmetric unit consists of a dimer with a molecular weight of ~ 46,000