Towards Automating Protein Structure Determination from NMR Data

Nuclear magnetic resonance (NMR) spectroscopy technique is becoming exceedingly significant due to its capability of studying protein structures in solution. However, NMR protein structure determination has remained a laborious and costly process until now, even with the help of currently available computer programs. After the NMR spectra are collected, the main road blocks to the fully automated NMR protein structure determination are peak picking from noisy spectra, resonance assignment from imperfect peak lists, and structure calculation from incomplete assignment and ambiguous nuclear Overhauser enhancements (NOE) constraints. The goal of this dissertation is to propose error-tolerant and highly-efficient methods that work well on real and noisy data sets of NMR protein structure determination and the closely related protein structure prediction problems. One major contribution of this dissertation is to propose a fully automated NMR protein structure determination system, AMR, with emphasis on the parts that I contributed. AMR only requires an input set with six NMR spectra. We develop a novel peak picking method, PICKY, to solve the crucial but tricky peak picking problem. PICKY consists of a noise level estimation step, a component forming step, a singular value decomposition-based initial peak picking step, and a peak refinement step. The first systematic study on peak picking problem is conducted to test the performance of PICKY. An integer linear programming (ILP)-based resonance assignment method, IPASS, is then developed to handle the imperfect peak lists generated by PICKY. IPASS contains an error-tolerant spin system forming method and an ILP-based assignment method. The assignment generated by IPASS is fed into the structure calculation step, FALCON-NMR. FALCON-NMR has a threading module, an ab initio module, an all-atom refinement module, and an NOE constraints-based decoy selection module. The entire system, AMR, is successfully tested on four out of five real proteins with practical NMR spectra, and generates 1.25A, 1.49A, 0.67A, and 0.88A to the native reference structures, respectively. Another contribution of this dissertation is to propose novel ideas and methods to solve three protein structure prediction problems which are closely related to NMR protein structure determination. We develop a novel consensus contact prediction method, which is able to eliminate server correlations, to solve the protein inter-residue contact prediction problem. We also propose an ultra-fast side chain packing method, which only uses local backbone information, to solve the protein side chain packing problem. Finally, two complementary local quality assessment methods are proposed to solve the local quality prediction problem for comparative modeling-based protein structure prediction methods

Computational method development for drug discovery

Protein-small molecule interactions play a central role in various aspects of the structural and functional organization of the cell and are therefore integral for drug discovery. The most comprehensive structural characterization of small molecule binding sites is provided by X-ray crystallography. However, it is often time-consuming and challenging to perform direct experimental analysis. Therefore, it is necessary to have computational methods that can predict binding site locations on unbound structures with accuracy close to that provided by X-ray crystallography. This thesis details four projects which involve the development of a fragment benchmark set, evaluation of allosteric sites in G Protein-Coupled Receptors (GPCRs), computational modeling of binding pocket dynamics, and the development of an Application Program Interface (API) framework for High-Performance Computing (HPC) centers. The first project provides a benchmark set for testing hot spot identification methods, emphasizing application to fragment-based drug discovery. Using the solvent mapping server, FTMap, which finds small molecule binding hot spots on proteins, we compared our benchmark set to an existing benchmark set that with a different method of construction. The second project details the effort to identify allosteric binding sites on GPCRs. We demonstrate that FTMap successfully identifies structurally determined allosteric sites in bound crystal structures and unbound structures. The project was further expanded to evaluate the conservation of allosteric sites across different classes, families, and types of GPCRs. The third project provides a structure-based analysis of cryptic site openings. Cryptic sites are pockets formed in ligand-bound proteins but not observed in unbound protein structures. Through analysis of crystal structures supplemented by molecular dynamics (MD) with enhanced sampling techniques, it was shown that cryptic sites can be grouped into three types: 1) “genuine” cryptic sites, which do not form without ligand binding, 2) spontaneously forming cryptic sites, and 3) cryptic sites impacted by mutations or off-site ligand binding. The fourth project presents an API framework for increasing the accessibility of HPC resources

Influence de la formulation sur la formation et la stabilisation des interfaces huile - eau dans les crèmes glacées

Les émulsifiants et les protéines participent à la création et à la stabilisation des différentes interfaces formées au cours de la fabrication des crèmes glacées qui fait évoluer le système d'une simple émulsion huile-dans-eau vers une mousse partiellement solide. La compréhension des mécanismes mis en jeu aux interfaces et la connaissance des propriétés des ingrédients sont nécessaires pour optimiser la fabrication et la stabilisation du produit fini. C'est dans ce contexte que différents systèmes, émulsion huile-dans-eau, mix de crème glacée et crèmes glacées ont été fabriqués à partir de quatre types de matières grasses différentes et stabilisés par deux types de mono- et diglycerides et deux sources protéiques. Des caractérisations multiples, telles que le profil thermique des matières grasses, le comportement rhéologique ou la réponse en fluorescence, appliquées à ces trois systèmes ont été utilisées. Il a été mis en évidence, dans les émulsions et dans les mixes, un phénomène compétitif aux interfaces entre les molécules tensioactives, principalement pendant la période de maturation. Ce phénomène est, en autre, influencé par le degré d'insaturation de l'émulsifiant et de la matière grasse. L'influence de la formulation a été également mise en évidence sur le produit fini, notamment en termes de comportement à la fonte et de perception organoleptique. Peu de corrélation entre les différentes techniques utilisées et entre les différents systèmes étudiés ont été observées suggérant la mise en place d'associations spécifiques entre ingrédients.The emulsifiers and the proteins take part in the creation and the stabilization of the various interfaces formed during the steps of ice cream processing which evolves from a simple oil-inwater emulsion to a partially solid foam. The aim of this study was to understand and control the mechanisms of action of the tensioactive molecules during the different steps of ice cream manufacture. In this context, various systems, oil-in-water emulsion, ice cream mix and ice cream, were manufactured based on four types of fat and stabilized by two types of mono- and diglyceride mixtures and two protein sources. Multiple of characterization, such as thermal behavior of fat, rheological parameters and fluorescence response, applied to these three systems were used. It was highlighted in the emulsions and mix systems that a competitive phenomenon at the oil-water interfaces occurred between emulsifiers and proteins especially during the ageing period. This phenomenon was influenced, among other things, by the degree of unsaturation of the emulsifier and of the fat. The influence of the formulation was also pointed out on the finished product in particular in term of melting behavior and organoleptic perception. Little correlation between the various techniques used and between the various systems studied was observed suggesting that specific associations between ingredients were taking place

Functional living biointerfaces to direct cell-material interaction

[EN] This thesis deals with the development of a living biointerface between synthetic substrates and living cells to engineer cell-material interactions for tissue engineering purposes. This living biointerface is made of Lactococcus lactis, a non-pathogenic lactic bacteria widely used as starter in the dairy industry and, recently, in the expression of heterologous proteins in applications such as oral vaccine delivery or membrane-bound expression of proteins. L. lactis has been engineered to display the III 7-10 fragment of the fibronectin fused to GFP as reporter protein. Fibronectin is a ubiquitous protein present in the extracellular matrix, a complex mesh of structural and adhesive proteins which serve as mechanical support and development niche for cells of a wide variety of tissues. This fragment contains two important sequences, RGD and PHSRN. RGD is an adhesive sequence that interacts with a wide range of integrins, membrane-bound receptors that play a role in cellular processes such as adhesion, migration, proliferation and differentiation. On the other hand, PHSRN binds synergistically with RGD to some integrins such as alpha-5-beta-1 and others, increasing the specificity of this interaction. Genetically engineered L. lactis has been thoroughly characterized to test its capabilities as a living interface. This strain was found to express the FNIII 7-10-GFP fragment covalently linked to the cell wall and biological activity and expression levels of this fragment was assessed with techniques such as Western blot, ELISA and immunofluorescence. Moreover, this strain still holds the ability to develop biofilms, communities of sessile, attached bacteria to abiotic surfaces which helps greatly in the generation of a stable monolayer of bacteria between synthetic substrates and mammalian cells. Mammalian cell behaviour in response to the expressed fibronectin fragment on L. lactis membrane was also assessed. Several cell lines were tested, such as Fn-/Fn- and NIH3T3 fibroblasts, C2C12 myoblasts and human bone-marrow derived mesenchymal cells. This living biointerface was found to trigger cell adhesion and FAK phosphorylation, a marker for intracellular integrin-mediated signalling in all of the tested cell lines. It also triggered myoblast-to-myotube differentiation on C2C12 cells. In hMSCs, the cell-wall exposed fibronectin fragment was found to enhance the phosphorylation of ERK1/2, a kinase involved in the MAPK pathway, which is deeply involved in a multitude of cellular processes related to differentiation, proliferation and migration. Nevertheless, this thesis is a proof of concept that this novel system can be further exploited to express almost any desired protein or small molecule to help in the development of new tissues from progenitor cells. These molecules can be either secreted in the medium or displayed in the membrane, and can also be constitutively expressed or in-demand, due to the great flexibility of L. lactis and the wide variety of expression systems available. This interface based on living bacteria establishes a new paradigm in surface functionalization for biomedical engineering applications.[ES] Esta tesis aborda el desarrollo de una biointerfase viviente entre materiales sintéticos y células vivas con el objetivo de dirigir la interacción célula-material en aplicaciones de ingeniería tisular. Esta biointerfase está compuesta de Lactococcus lactis, una bacteria láctica no patógena, ampliamente usada en la industria láctea como inóculo, y, recientemente, en la expresión heteróloga de proteínas para su uso como vacunas de administración oral o su expresión en membrana. L. lactis ha sido genéticamente modificado para expresar el fragmento III 7-10 de la fibronectina, unida a GFP como reporter. La fibronectina es una proteína presente de forma ubicua en la matriz extracelular, una compleja red de proteínas adhesivas y estructurales cuyo propósito es servir como soporte estructural y como nicho de desarrollo para diversos tejidos. Este fragmento contiene dos secuencias importantes, RGD y PHSRN. RGD es una secuencia adhesiva de unión que interacciona con una amplia variedad de integrinas, receptores de membrana que juegan muchos e importantes papeles en diferentes procesos celulares, como adhesión, proliferación, migración o diferenciación. Por otra parte, PHSRN se une a las integrinas de forma sinérgica con RGD facilitando aún más estos procesos y aumentando la especificidad de esta interacción. Esta cepa de L. lactis modificada ha sido ampliamente caracterizada para estudiar su idoneidad como interfaz funcional viviente. Se ha demostrado que L. lactis es capaz de expresar el fragmento FNIII7-10-GFP covalentemente anclado a la pared celular bacteriana, habiéndose caracterizado también su actividad biológica con técnicas como Western blot, ELISA e inmunofluorescencia. Esta cepa mantiene la capacidad de desarrollo de biofilms presente en la gran mayoría de microorganismos. Los biofilms son comunidades de bacterias sésiles adheridas a un sustrato que pueden ser usadas como interfase física entre células de mamífero y sustratos abióticos. También se ha estudiado la respuesta celular a la fibronectina expuesta en la membrana de L. lactis. Se estudiaron varias líneas celulares, como fibroblastos Fn-/Fn- y NIH3T3, mioblastos C2C12 y células mesenquimales humanas derivadas de médula ósea. Esta interfase viviente fue capaz de provocar respuesta celular en forma de adhesión en todas las líneas estudiadas, además de inducir diferenciación de mioblastos a miotubos en C2C12 y de provocar la fosforilación de FAK, un marcador de señalización celular mediada por integrinas. En células mesenquimales humanas se demostró la capacidad del fragmento de fibronectina expuesto para fosforilar ERK1/2, una kinasa perteneciente a la ruta de señalización MAPK, ruta que forma parte de muchos procesos celulares importantes como diferenciación, proliferación y migración. Pese a todo, esta tesis es sólo una prueba de concepto de un sistema que puede ser utilizado para expresar casi cualquier proteína o molécula pequeña deseada, que puede ser muy útil en el desarrollo de nuevos tejidos a partir de sus células progenitoras. Estas moléculas pueden ser secretadas en el medio o ancladas en la pared celular, de forma constitutiva o bajo demanda, debido a la flexibilidad y amplia variedad de sistemas de expresión disponibles para L. lactis. Esta biointerfase basada en bacterias vivas establece un nuevo paradigma en el campo de la funcionalización de superficies para aplicaciones de ingeniería biomédica.[CA] Aquesta tesi aborda el desenvolupament d'una interfase viva entre materials sintètics i cèl·lules vives amb l'objectiu de dirigir la interacció cèl·lula-material, per al seu ús en aplicacions d'enginyeria tissular. Aquesta interfase està composta de Lactococcus lactis, un bacteri làctic, no patogènic i àmpliament utilitzat en l'industria làctica com a inòcul, i, recentment, en l'expressió heteròloga de proteïnes per al seu ús com vacunes d'administració oral o per a la seva expressió en membrana. L. lactis ha sigut genèticament modificada per a expressar el fragment III7-10 de la fibronectina, unida a GFP com a reporter. La fibronectina és una proteïna present de forma ubiqua en la matriu extracel·lular, una complexa xarxa de proteïnes adhesives i estructurals que s'utilitzen com a suport estructural i com a nínxol de desenvolupament per a diversos teixits. Aquest fragment conté dos seqüències importants, RGD i PHSRN. RGD és una seqüència adhesiva d'unió a integrines, receptors de membrana que juguen molts i molt importants papers en diferents processos cel·lulars, com poden ser adhesió, proliferació, migració o diferenciació. Per altra banda, PHSRN s'uneix a les integrines de forma sinèrgica amb RGD facilitant encara més aquests processos i augmentant l'especificitat d'aquesta interacció. Aquesta modificació genètica de L. lactis ha estat àmpliament caracteritzada per provar les seves característiques com a interfase funcional vivent. S'ha demostrat que L. lactis és capaç d'expressar el fragment FNIII 7-10-GFP covalentment ancorat a la paret cel·lular bacteriana, havent-se caracteritzat també la seva activitat biològica amb tècniques com Western blot, ELISA i immunofluorescència. A més, aquest cep manté la capacitat de desenvolupament de biofilms, comunitats de bacteris sèssils adherits a un substrat que poden ser utilitzades com a interfase física entre cèl·lules de mamífer i substrats abiòtics. També s'ha estudiat la resposta cel·lular a la fibronectina expressada en la paret cel·lular de L. lactis. El estudi es va fer utilitzant diverses línies cel·lulars, com fibroblasts Fn-/Fn- i NIH3T3, mioblasts C2C12 i cèl·lules mesenquimals humanes derivades de medul·la òssia. Aquesta interfase vivent va ser capaç de provocar resposta cel·lular en forma d'adhesió a totes les línies estudiades, a més d'induir diferenciació de mioblasts a miotubs en C2C12 i de provocar la fosforilació de FAK, un marcador de senyalització cel·lular mediat per integrines, en les línies assajades. En cèl·lules mesenquimals humanes es va demostrar la capacitat del fragment de fibronectina exposat per fosforilar ERK1/2, una kinasa pertanyent a la ruta de senyalització MAPK, ruta que forma part de molts processos cel·lulars importants com diferenciació, proliferació i migració. Malgrat tot, aquesta tesi mostra només una prova de concepte d'un sistema que pot ser utilitzat per expressar gairebé qualsevol proteïna o molècula petita desitjada, que pot ser molt útil en el desenvolupament de nous teixits a partir de les seves cèl·lules progenitores. Aquestes molècules poden ser secretades en el medi o ancorades a la paret cel·lular, de manera constitutiva o sota demanda, a causa de la flexibilitat i àmplia varietat de sistemes d'expressió disponibles per L. lactis Aquesta biointerfase basada en bacteris vius estableix un nou paradigma en el camp de la funcionalització de superfícies per a aplicacions d'enginyería biomèdica.Rodrigo Navarro, A. (2015). Functional living biointerfaces to direct cell-material interaction [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51461TESI

Optimising the statistical pipeline for quantitative proteomics

Background Label-free quantitative proteomics utilises differential expression (DE) analysis of high-throughput methods for mass spectrometry, providing insight into disease biomarkers, protein involvement in metabolic pathways or facilitating drug discovery. Applying statistical techniques to assess the significance of proteins changing in abundance is complicated by the properties of the data. Small numbers of samples containing vast numbers of features result in large sample-to-sample variation where the comparison of means can be distorted by outliers. Limitations of benchmarking data and the complexity of the algorithms make software comparison challenging. Full optimisation of the proteomics workflow is difficult, and it is a daunting task for the biologist to intuitively obtain optimal results. The aim of this Industrial CASE PhD studentship, in collaboration with Nonlinear Dynamics, the developers of Progenesis QI for Proteomics (QIP), is to provide an improved statistical pipeline that could be implemented in the Progenesis QIP workflow. Methods Benchmarking of three existing statistical approaches: QPROT, ANOVA as implemented directly in Progenesis QIP, and MSstats, was conducted traditionally, using spike-in datasets, and through the implementation of a novel method, using biological data and applying pathway analysis as an evaluation metric. Normalisation methods and the optimal threshold for defining significance were also investigated. Following this, an optimised proteomics pipeline was developed and implemented using high performance computing cluster for parallelisation of multiple combinations of methods for DE analysis, normalisation, and significance threshold selection. Functional enrichment analysis of proteins defined as changing was used to assess the results and the optimal parameter combination returned to the user. Effectiveness of this approach was demonstrated by comparing the best results from the pipeline with enrichment analysis of the output from the current Progenesis QIP workflow. Results Overall, the results of benchmarking gave no consensus on best method for DE, normalisation method, or significance threshold and the correct combination of parameters appeared to be dependent on the characteristics of the individual datasets. The results also showed that the choice of an appropriate normalisation method is an important and underappreciated factor in differential expression analysis and that the optimal threshold for defining significance varied greatly from the generally accepted value of p < 0.05. The optimised pipeline’s performance was superior to a standard analysis using Progenesis QIP. To our knowledge, this is the only end-to-end pathway analysis pipeline designed for proteomics data, enabling users to iterate through multiple options for finding the best normalisation method and the best significance threshold for pathway analysis