Analysis of Three-Dimensional Protein Images

A fundamental goal of research in molecular biology is to understand protein structure. Protein crystallography is currently the most successful method for determining the three-dimensional (3D) conformation of a protein, yet it remains labor intensive and relies on an expert's ability to derive and evaluate a protein scene model. In this paper, the problem of protein structure determination is formulated as an exercise in scene analysis. A computational methodology is presented in which a 3D image of a protein is segmented into a graph of critical points. Bayesian and certainty factor approaches are described and used to analyze critical point graphs and identify meaningful substructures, such as alpha-helices and beta-sheets. Results of applying the methodologies to protein images at low and medium resolution are reported. The research is related to approaches to representation, segmentation and classification in vision, as well as to top-down approaches to protein structure prediction.Comment: See http://www.jair.org/ for any accompanying file

Representation, searching and discovery of patterns of bases in complex RNA structures

We describe a graph theoretic method designed to perform efficient searches for substructural patterns in nucleic acid structural coordinate databases using a simplified vectorial representation. Two vectors represent each nucleic acid base and the relative positions of bases with respect to one another are described in terms of distances between the defined start and end points of the vectors on each base. These points comprise the nodes and the distances the edges of a graph, and a pattern search can then be performed using a subgraph isomorphism algorithm. The minimal representation was designed to facilitate searches for complex patterns but was first tested on simple, well-characterised arrangements of bases such as base pairs and GNRA-tetraloop receptor interactions. The method performed very well for these interaction types. A survey of side-by-side base interactions, of which the adenosine platform is the best known example, also locates examples of similar base rearrangements that we consider to be important in structural regulation. A number of examples were found, with GU platforms being particularly prevalent. A GC platform in the RNA of the Thermus thermophilus small ribosomal subunit is in an analogous position to an adenosine platform in other species. An unusual GG platform is also observed close to one of the substrate binding sites in Haloarcula marismortui large ribosomal subunit RNA

Horizontal Visibility graphs generated by type-I intermittency

The type-I intermittency route to (or out of) chaos is investigated within the Horizontal Visibility graph theory. For that purpose, we address the trajectories generated by unimodal maps close to an inverse tangent bifurcation and construct, according to the Horizontal Visibility algorithm, their associated graphs. We show how the alternation of laminar episodes and chaotic bursts has a fingerprint in the resulting graph structure. Accordingly, we derive a phenomenological theory that predicts quantitative values of several network parameters. In particular, we predict that the characteristic power law scaling of the mean length of laminar trend sizes is fully inherited in the variance of the graph degree distribution, in good agreement with the numerics. We also report numerical evidence on how the characteristic power-law scaling of the Lyapunov exponent as a function of the distance to the tangent bifurcation is inherited in the graph by an analogous scaling of the block entropy over the degree distribution. Furthermore, we are able to recast the full set of HV graphs generated by intermittent dynamics into a renormalization group framework, where the fixed points of its graph-theoretical RG flow account for the different types of dynamics. We also establish that the nontrivial fixed point of this flow coincides with the tangency condition and that the corresponding invariant graph exhibit extremal entropic properties.Comment: 8 figure

EPI-001, A Compound Active against Castration-Resistant Prostate Cancer, Targets Transactivation Unit 5 of the Androgen Receptor

ACKNOWLEDGEMENTS We thank J. M. Valverde (IRB) as well as the NMR facilities of the University of Barcelona (CCiT UB) and the Instituto de Química Física Rocasolano (IQFR, CSIC) for their assistance in, respectively, protein production and NMR. This work was supported by IRB, ICREA (X.S.), Obra Social “la Caixa” (Fellowship to E.D.M. and CancerTec grants to X.S.) MICINN (CTQ2009-08850 to X.S.), MINECO (BIO2012-31043 to X.S.; CTQ2014-56361-P to A.R), Marató de TV3 (102030 to X.S. and 102031 to E.E.P) the COFUND programme of the European Commission (C.T.W.P., A. R. and X.S.), the European Research Council (CONCERT, contract number 648201, to X.S.), the Ramón y Cajal program of MICINN (RYC-2011-07873 to C.W.B.) the Serra Hunter Programme (E.E.P.) and AGAUR (SGR-2014-56RR14 to E.E.P). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain)Peer reviewedPostprin

A Hydrophobic Gate in an Ion Channel: The Closed State of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is the prototypic member of the `Cys-loop' superfamily of ligand-gated ion channels which mediate synaptic neurotransmission, and whose other members include receptors for glycine, gamma-aminobutyric acid, and serotonin. Cryo-electron microscopy has yielded a three dimensional structure of the nAChR in its closed state. However, the exact nature and location of the channel gate remains uncertain. Although the transmembrane pore is constricted close to its center, it is not completely occluded. Rather, the pore has a central hydrophobic zone of radius about 3 A. Model calculations suggest that such a constriction may form a hydrophobic gate, preventing movement of ions through a channel. We present a detailed and quantitative simulation study of the hydrophobic gating model of the nicotinic receptor, in order to fully evaluate this hypothesis. We demonstrate that the hydrophobic constriction of the nAChR pore indeed forms a closed gate. Potential of mean force (PMF) calculations reveal that the constriction presents a barrier of height ca. 10 kT to the permeation of sodium ions, placing an upper bound on the closed channel conductance of 0.3 pS. Thus, a 3 A radius hydrophobic pore can form a functional barrier to the permeation of a 1 A radius Na+ ion. Using a united atom force field for the protein instead of an all atom one retains the qualitative features but results in differing conductances, showing that the PMF is sensitive to the detailed molecular interactions.Comment: Accepted by Physical Biology; includes a supplement and a supplementary mpeg movie can be found at http://sbcb.bioch.ox.ac.uk/oliver/download/Movies/watergate.mp

NMR analysis of synthetic human serum albumin alpha-helix 28 identifies structural distortion upon amadori modification

The non-enzymatic reaction between reducing sugars and long-lived proteins in vivo results in the formation of glycation and advanced glycation end products, which alter the properties of proteins including charge, helicity, and their tendency to aggregate. Such protein modifications are linked with various pathologies associated with the general aging process such as Alzheimer disease and the long-term complications of diabetes. Although it has been suggested that glycation and advanced glycation end products altered protein structure and helicity, little structural data and information currently exist on whether or not glycation does indeed influence or change local protein secondary structure. We have addressed this problem using a model helical peptide system containing a di-lysine motif derived from human serum albumin. We have shown that, in the presence of 50 mM glucose and at 37 degrees C, one of the lysine residues in the di-lysine motif within this peptide is preferentially glycated. Using NMR analysis, we have confirmed that the synthetic peptide constituting this helix does indeed form a alpha-helix in solution in the presence of 30% trifluoroethanol. Glycation of the model peptide resulted in the distortion of the alpha-helix, forcing the region of the helix around the site of glycation to adopt a 3(10) helical structure. This is the first reported evidence that glycation can influence or change local protein secondary structure. The implications and biological significance of such structural changes on protein function are discussed

The role of dynamic hydrogen bond networks in protonation coupled dynamics of retinal proteins

Hydrogen bonds (H-bonds) are an essential interaction in membrane proteins. Embedded in complex hydrated lipid bilayers, intramolecular interactions through the means of hydrogen bonding networks are often crucial for the function of the protein. Internal water molecules that occupy stable sites inside the protein, or water molecules that visit transiently from the bulk, can play an important role in shaping local conformational dynamics forming complex networks that bridge regions of the protein via water-mediated hydrogen bonds that can function as wires for the transferring of protons as a part of the protein’s function. For example, the membrane-embedded channelrhodopsins which are found in archaea are proteins that couple light induced isomerization of a retinal chromophore with proton transfer reactions and passive flow of cations through their pore. I contributed to the development of a new algorithm package that features a unique approach to H-bond analyses. I performed analyses of long Molecular Dynamics (MD) trajectories of channelrhodopsin variants embedded in hydrated lipid membranes and large data sets of static structures, to detect and dissect dynamic hydrogen-bond networks. The photocycle of channelrhodopsins begins with absorption and isomerization of the retinal from an all-trans state to a 13-cis state and followed by the deprotonation of the Schiff base. Thus, the retinal is found in the epicenter of the analyses. Through the use of 2-dimensional graphs of the protein H-bond networks I identified protein groups potentially important for the proton transfer activity. Local dynamics are highly affected by point mutations of amino acids important for function. The interior of channelrhodopsin C1C2 hosts extensive networks of protein and H-bonded-water molecules, and a never reported before, network that can bridge transiently the two retinal chromophores in channelrhodopsin dimers. In a recently identified inward proton pump, AntR, I applied centrality measures on MD trajectories of the homology model I generated, to assess the communication of the amino acid residues within the networks. I detected a frequently sampled long water chain that connects the retinal with a candidate proton acceptor, as well as a conserved serine in the vicinity of the retinal chromophore plays a significant role in the connectivity and communication of the H-bond networks upon isomerization. A similar water bridge is sampled in independent simulations of ChR2, where a participant for the proton donor group connects to the 13-cis,15-anti retinal. Proton transfer reactions often take place through certain amino acids, forming patterns. I analyzed H-bond patterns or motifs in large hand-curated datasets of static structures of α-transmembrane helix proteins, organized according to the superfamilies they belong, their function and an alternative classification method. The presence of motifs in TM proteins is tightly related to their families/superfamilies of the host protein and their position along the membrane normal.Wasserstoffbrücken (H-Brücke) sind eine wesentliche Wechselwirkung in Membranproteinen. Eingebettet in komplexe hydratisierte Lipiddoppelschichten sind intramolekulare Wechselwirkungen über Wasserstoffbrückenbindungsnetzwerke oft entscheidend für die Funktion des Proteins. Interne Wassermoleküle, die stabile Stellen im Inneren des Proteins besetzen, oder Wassermoleküle, die vorübergehend aus der Masse zu Besuch kommen, können eine wichtige Rolle bei der Gestaltung der lokalen Konformationsdynamik spielen, indem sie komplexe Netzwerke bilden, die Regionen des Proteins über wasservermittelte Wasserstoffbrückenbindungen überbrücken, die als Drähte für den Transfer von Protonen als Teil der Proteinfunktion funktionieren können. Die in Archaeen vorkommenden, in die Membran eingebetteten Kanalrhodopsine sind beispielsweise Proteine, die die lichtinduzierte Isomerisierung eines Retinachromophors mit Protonentransferreaktionen und dem passiven Fluss von Kationen durch ihre Pore verbinden. Ich habe an der Entwicklung eines neuen Algorithmuspakets mitgewirkt, das einen einzigartigen Ansatz für H-Bindungsanalysen bietet. Ich habe lange Molekulardynamik-Trajektorien von Kanalrhodopsine-Varianten, die in hydratisierte Lipidmembranen eingebettet sind, sowie große Datensätze statischer Strukturen analysiert, um dynamische Wasserstoffbrücken-bindungsnetzwerke zu erkennen und zu zerlegen. Der Photozyklus der Kanalrhodopsine beginnt mit der Absorption und Isomerisierung des Retinals von einem all-trans-Zustand zu einem 13-cis-Zustand, gefolgt von der Deprotonierung der Schiff-Base. Somit steht das Retinal im Mittelpunkt der Analysen. Durch die Verwendung von 2-dimensionalen Graphen der Protein- H-Brückenetzwerke identifizierte ich Proteingruppen, die für die Protonentransferaktivität wichtig sein könnten. Die lokale Dynamik wird durch Punktmutationen der für die Funktion wichtigen Aminosäuren stark beeinflusst. Das Innere von Kanalrhodopsine C1C2 beherbergt ausgedehnte Netzwerke von Protein- und H-Brücke-Wassermolekülen und ein bisher unbekanntes Netzwerk, das die beiden retinalen Chromophore in Kanalrhodopsine-Dimeren vorübergehend überbrücken kann. In einer kürzlich identifizierten Protonenpumpe, AntR, wendete ich Zentralitätsmaße auf MD-Trajektorien des von mir erstellten Homologiemodells an, um die Kommunikation der Aminosäurereste innerhalb der Netzwerke zu bewerten. Ich fand, dass eine häufig gesampelte lange Wasserkette, die das Retinal mit einem Protonenakzeptor verbindet, sowie ein konserviertes Serin in der Nähe des Retinal-Chromophors eine wichtige Rolle bei der Konnektivität und Kommunikation der H-Brückesnetzwerke bei der Isomerisierung spielt. Eine ähnliche Wasserbrücke ist in unabhängigen Simulationen von Kanalrhodopsine-2 zu finden, wo ein Teilnehmer für die Protonendonorgruppe mit dem 13-cis,15-anti-Retinal verbunden ist. Protonenübertragungsreaktionen finden oft über bestimmte Aminosäuren statt und bilden Muster. Ich analysierte H-Brückemuster oder -motive in großen, von Hand kuratierten Datensätzen statischer Strukturen von α-Transmembranhelix-Proteinen, geordnet nach den Superfamilien, zu denen sie gehören, ihrer Funktion und einer alternativen Klassifizierungsmethode. Das Vorhandensein von Motiven in TM-Proteinen steht in engem Zusammenhang mit ihren Familien/Superfamilien des Wirtsproteins und ihrer Position entlang der Membrannormale