Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Studies of Protein-Protein and Protein-Water Interactions by Small Angle X-Ray Scattering, Terahertz Spectroscopy, ASMOS, And Computer Simulation

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 µm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.

Exploring protein-RNA interactions with site-directed mutagenesis and phage display

The importance of RNA as a mediator of genetic information is widely appreciated. RNA molecules also participate in the regulation of various post-transcriptional activities, such as mRNA splicing, editing, RNA stability and transport. Their regulatory roles for these activities are highly dependent on finely tuned associations with cognate proteins. The RNA recognition motif (RRM) is an ancient RNA binding module that participates in hundreds of essential activities where specific RNA recognition is required. We have applied phage display and site-directed mutagenesis to dissect principles of RRM-controlled RNA recognition. The model systems we are investigating are U1A and CUG-BP1. In this dissertation, the molecular basis of the binding affinity of U1A-RNA beyond individual contacts was investigated. We have identified and evaluated the contributions of the local cooperativity formed by three neighboring residues (Asn15, Asn16 and Glu19) to the stability of the U1A-RNA complex. The localized cooperative network was mapped by double-mutant cycles and explored using phage display. We also showed that a cluster of these residues forms a “hot spot” on the surface of U1A; a single substitution at position 19 with Gln or His can alter the binding properties of U1A to recognize a non-cognate G4U RNA. Finally, we applied a deletion analysis of CUG-BP1 to define the contributions of individual RRMs and RRM combinations to the stability of the complex formed between CUG-BP1 and the GRE sequence. The preliminary results showed RRM3 of CUG-BP1 is a key domain for RNA binding. It possibly binds to the GRE sequence cooperatively with RRM2 of CUG-BP1. RRM1 of CUG-BP1 is not required for GRE recognition, but may be important for maintaining the stability of the full-length CUG-BP1.

CCTα

In the cell, the correct folding of many proteins depends on the function of preexisting ones known as Molecular Chaperones (for a review see Hartl and Hayer-Hartl 2009). These, were defined as proteins that bind to and stabilize an otherwise unstable conformation of another protein, and by controlling binding and release, facilitate its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation. Molecular chaperones do not convey steric information specifying correct folding: instead, they prevent incorrect interactions within and between nonnative peptides, thus typically increasing the yield but not the rate of folding reactions. Molecular chaperones are ubiquitous and comprise several protein families that are structurally unrelated (Hartl and Hayer-Hartl 2009). The Hsp70s and the Chaperonin families have been extensively studied.

Exploring unfrequent events with accelerated molecular dynamics simulations: from photochemistry to drug design

La meva incorporació al grup de recerca del Prof. McCammon (University of California San Diego) en qualitat d’investigador post doctoral amb una beca Beatriu de Pinós, va tenir lloc el passat 1 de desembre de 2010; on vaig dur a terme les meves tasques de recerca fins al darrer 1 d’abril de 2012. El Prof. McCammon és un referent mundial en l’aplicació de simulacions de dinàmica molecular (MD) en sistemes biològics d’interès humà. La contribució més important del Prof. McCammon en la simulació de sistemes biològics és el desenvolupament del mètode de dinàmiques moleculars accelerades (AMD). Les simulacions MD convencionals, les quals estan limitades a l’escala de temps del nanosegon (~10-9s), no son adients per l’estudi de sistemes biològics rellevants a escales de temps mes llargues (μs, ms...). AMD permet explorar fenòmens moleculars poc freqüents però que son clau per l’enteniment de molts sistemes biològics; fenòmens que no podrien ser observats d’un altre manera. Durant la meva estada a la “University of California San Diego”, vaig treballar en diferent aplicacions de les simulacions AMD, incloent fotoquímica i disseny de fàrmacs per ordinador. Concretament, primer vaig desenvolupar amb èxit una combinació dels mètodes AMD i simulacions Car-Parrinello per millorar l’exploració de camins de desactivació (interseccions còniques) en reaccions químiques fotoactivades. En segon lloc, vaig aplicar tècniques estadístiques (Replica Exchange) amb AMD en la descripció d’interaccions proteïna-lligand. Finalment, vaig dur a terme un estudi de disseny de fàrmacs per ordinador en la proteïna-G Rho (involucrada en el desenvolupament de càncer humà) combinant anàlisis estructurals i simulacions AMD. Els projectes en els quals he participat han estat publicats (o estan encara en procés de revisió) en diferents revistes científiques, i han estat presentats en diferents congressos internacionals. La memòria inclosa a continuació conté més detalls de cada projecte esmentat.

Mapeamento das interações proteína-ligante através de técnicas de RMN de ¹H utilizando detecção do ligante

NMR is a valuable screening tool for the binding of ligands to proteins providing structural information on both protein and ligands and is thus largely applied to drug-discovery. Among the recent NMR techniques to probe weak binding protein-ligand complexes we have critically evaluated the advantages and disadvantages of STD (Saturation Transfer Difference), WaterLOGSY (Water Ligand Observation with Gradient Spectroscopy), NOE pumping and DOSY-NOESY (Diffusion-Ordered NOESY) using a mixture of BSA (bovine serum albumin) plus salicylic acid, caffeine, citric acid, adipic acid and D-glucose.

Immersive Projection for biochemical pattern matching

In all biological processes, protein molecules and other small molecules interact to function and form transient macromolecular complexes. This interaction of two or more molecules can be described by a docking event. Docking is an important phase for structure-based drug design strategies, as it can be used as a method to simulate protein-ligand interactions. Various docking programs exist that allow automated docking, but most of them have limited visualization and user interaction. It would be advantageous if scientists could visualize the molecules participating in the docking process, manipulate their structures and manually dock them before submitting the new conformations to an automated docking process in an immersive environment, which can help stimulate the design/docking process. This also could greatly reduce docking time and resources. To achieve this, we propose a new virtual modelling/docking program, whereby the advantages of virtual modelling programs and the efficiency of the algorithms in existing docking programs will be merged.

Determinação do grau de ionização de aminoácidos polares carregados

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão, purificação e caracterização de proteínas dos fitovírus SBMV e RSPaV

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kontaktmechanik und Strukturierung von festkörperunterstützen Lipidmembranen

Die vorliegende Arbeit beschreibt unter anderem die Realisierung eines Assays aus mikrostrukturierten und selektiv funktionalisierten künstlichen Membransegmenten auf einem Chip. Die Strukturierungsmethode kombiniert die softlithographische Technik des Mikroformens in Kapillaren mit der Vesikelspreittechnik und bietet ein elegantes Verfahren, einzeln adressierbare Lipidsegmente im Mikrometer Regime zu erzeugen. Unter Berücksichtigung des hydrodynamischen Fließverhaltens und der Stabilitätskriterien für PDMS-Elastomere wurden außerdem neue Strukturen entwi-ckelt, die für den kombinierten Einsatz von Rasterkraftmikroskopie und Fluoreszenz-mikroskopie optimiert sind. Die Anwendbarkeit des Lab-On-A-Chip-Devices als Bio-sensor wurde durch zwei prominente Protein-Rezeptor-Bindungsstudien fluores-zenzmikroskopisch und rasterkraftmikroskopisch belegt. Im zweiten Teil der Arbeit sind die mechanischen und adhäsiven Eigenschaften aus-gewählter Lipidsysteme mit einer neuen Charakterisierungstechnik untersucht wor-den, die die Kontaktmechanik von Rastersonden und Lipidmembranen auf Basis der Digitalisierung von Hochgeschwindigkeitskraftkurven und einer automatisierten Multi-parameteranalyse quantitativ erfasst. Dabei konnte die Korrelation zwischen der Ad-häsion und den materialspezifischen Durchbruchlängen und Durchbruchkräften, die charakteristische Stabilitätsparameter der Lipidmembran darstellen, auf Systemen mit variierenden Kopfgruppen und Kettenlängen analysiert werden. Das Verfahren erlaubte zudem die simultane Quantifizierung der elastischen Eigenschaften der Li-piddoppelschichten. Zu den Kraftkurven wurden Simulationen der Systemantwort durchgeführt, die ein tieferes Verständnis der Kontrastentstehung ermöglichen.

Mechanisms of Ligand–Protein Interaction in Sec-14-like Transporters Investigated by Computer Simulations

We review our recent work on protein-ligand interactions in vitamin transporters of the Sec-14-like protein. Our studies focused on the cellular-retinaldehyde binding protein (CRALBP) and the alpha-tocopherol transfer protein (alpha-TTP). CRALBP is responsible for mobilisation and photo-protection of short-chain cis-retinoids in the dim-light visual cycle or rod photoreceptors. alpha-TTP is a key protein responsible for selection and retention of RRR-alpha-tocopherol, the most active isoform of vitamin E in superior animals. Our simulation studies evidence how subtle chemical variations in the substrate can lead to significant distortion in the structure of the complex, and how these changes can either lead to new protein function, or be used to model engineered protein variants with tailored properties. Finally, we show how integration of computational and experimental results can contribute in synergy to the understanding of fundamental processes at the biomolecular scale.

Mapping protein-protein interactions by affinity-directed mass spectrometry.

A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.

Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.

Flavonoid interactions with human transthyretin: Combined structural and thermodynamic analysis

Transthyretin (TTR) is a carrier protein involved in human amyloidosis. The development of small molecules that may act as TTR amyloid inhibitors is a promising strategy to treat these pathologies. Here we selected and characterized the interaction of flavonoids with the wild type and the V30M amyloidogenic mutant TTR. TTR acid aggregation was evaluated in vitro in the presence of the different flavonoids. The best TTR aggregation inhibitors were studied by Isothermal Titration Calorimetry (ITC) in order to reveal their thermodynamic signature of binding to TTRwt. Crystal structures of TTRwt in complex with the top binders were also obtained, enabling us to in depth inspect TTR interactions with these flavonoids. The results indicate that changing the number and position of hydroxyl groups attached to the flavonoid core strongly influence flavonoid recognition by TTR, either by changing ligand affinity or its mechanism of interaction with the two sites of TTR. We also compared the results obtained for ITRwt with the V30M mutant structure in the apo form, allowing us to pinpoint structural features that may facilitate or hamper ligand binding to the V30M mutant. Our data show that the TTRwt binding site is labile and, in particular, the central region of the cavity is sensible for the small differences in the ligands tested and can be influenced by the Met30 amyloidogenic mutation, therefore playing important roles in flavonoid binding affinity, mechanism and mutant protein ligand binding specificities. (C) 2012 Elsevier Inc. All rights reserved.