Pervasive, conserved secondary structure in highly charged protein regions

Understanding how protein sequences confer function remains a defining challenge in molecular biology. Two approaches have yielded enormous insight yet are often pursued separately: structure-based, where sequence-encoded structures mediate function, and disorder-based, where sequences dictate physicochemical and dynamical properties which determine function in the absence of stable structure. Here we study highly charged protein regions (>40% charged residues), which are routinely presumed to be disordered. Using recent advances in structure prediction and experimental structures, we show that roughly 40% of these regions form well-structured helices. Features often used to predict disorder—high charge density, low hydrophobicity, low sequence complexity, and evolutionarily varying length—are also compatible with solvated, variable-length helices. We show that a simple composition classifier predicts the existence of structure far better than well-established heuristics based on charge and hydropathy. We show that helical structure is more prevalent than previously appreciated in highly charged regions of diverse proteomes and characterize the conservation of highly charged regions. Our results underscore the importance of integrating, rather than choosing between, structure- and disorder-based approaches

The Use of Artificial Intelligence Techniques for Protein Structure Prediction

The conventional technique for computerized protein structure prediction uses several programming languages such as Fortran, C, Pascal etc. With recent advances in programming languages and the development of rule-based systems, the computerized part of the problem is undergoing major change. This thesis sets out the idea of extending the properties of an intelligent rule-based system and recognising incomplete nature of knowledge for this problem. It reviews the existing architectures and characteristics that embody an intelligent system. As the outcome of the idea, a new system called PREDMOLL, written in Prolog, is developed. PREDMOLL is based on the blackboard architecture with several other extra features. This thesis also reviews some current uncertainty techniques and developes a formula based on a modifications of the Bayes theorem, to deal with multiple hypotheses. The problem of conditional independence assumption is reduced to the minimum. The formula is used as a decision-making criterion to determine secondary structure boundaries. For tertiary structure prediction, this thesis suggests a similarity value for primary sequence homology to overcome the problem of arbitrary uncertainty values in rules. PREDMOLL and the uncertainty techniques incorporated with it are used to test the hypothesis that the performance of protein structure prediction is improved by combining several methods. The test is carried out by a series of experimental predictions with user-defined rules and predefined constraints. The behaviour of PREDMOLL during the problemsolving process of the experiments is shown. The results obtained yield improvements in precision for secondary structure prediction and further improvements are expected. For tertiary structure prediction, some preliminary progress is shown and, due to lack of genuine rules, ad-hoc rules are generated from the protein data base. The status of PREDMOLL and its advantages over other systems is discussed. Several suggestions are made to improve current facilities in PREDMOLL and problems in a wider domain. Suggestions are also made for further improvements in tertiary structure prediction

Residue contacts predicted by evolutionary covariance extend the application of ab initio molecular replacement to larger and more challenging protein folds

For many protein families, the deluge of new sequence information together with new statistical protocols now allow the accurate prediction of contacting residues from sequence information alone. This offers the possibility of more accurate ab initio (non-homology-based) structure prediction. Such models can be used in structure solution by molecular replacement (MR) where the target fold is novel or is only distantly related to known structures. Here, AMPLE, an MR pipeline that assembles search-model ensembles from ab initio structure predictions (`decoys'), is employed to assess the value of contact-assisted ab initio models to the crystallographer. It is demonstrated that evolutionary covariance-derived residue–residue contact predictions improve the quality of ab initio models and, consequently, the success rate of MR using search models derived from them. For targets containing β-structure, decoy quality and MR performance were further improved by the use of a β-strand contact-filtering protocol. Such contact-guided decoys achieved 14 structure solutions from 21 attempted protein targets, compared with nine for simple Rosetta decoys. Previously encountered limitations were superseded in two key respects. Firstly, much larger targets of up to 221 residues in length were solved, which is far larger than the previously benchmarked threshold of 120 residues. Secondly, contact-guided decoys significantly improved success with β-sheet-rich proteins. Overall, the improved performance of contact-guided decoys suggests that MR is now applicable to a significantly wider range of protein targets than were previously tractable, and points to a direct benefit to structural biology from the recent remarkable advances in sequencing

A graph neural network approach to automated model building in cryo-EM maps

Electron cryo-microscopy (cryo-EM) produces three-dimensional (3D) maps of the electrostatic potential of biological macromolecules, including proteins. Along with knowledge about the imaged molecules, cryo-EM maps allow de novo atomic modelling, which is typically done through a laborious manual process. Taking inspiration from recent advances in machine learning applications to protein structure prediction, we propose a graph neural network (GNN) approach for automated model building of proteins in cryo-EM maps. The GNN acts on a graph with nodes assigned to individual amino acids and edges representing the protein chain. Combining information from the voxel-based cryo-EM data, the amino acid sequence data and prior knowledge about protein geometries, the GNN refines the geometry of the protein chain and classifies the amino acids for each of its nodes. Application to 28 test cases shows that our approach outperforms the state-of-the-art and approximates manual building for cryo-EM maps with resolutions better than 3.5 \r{A}

Modelling the structural, functional and phenotypic consequences of protein coding mutations

Proteins are integral to all cellular processes and underpin the function of all extant organisms, meaning variants impacting them are a primary cause of phenotypic variation. Protein coding variants are a key area of study in biology, with relevance from structural and molecular biology to population genetics. They are also medically important, impacting inherited genetic diseases, cancer and response to pathogens. Recent advances in highthroughput experimental techniques have opened the door to many new approaches in biology, and protein variants are no exception. Deep mutational scanning experiments exhaustively measure the fitness of variants in a protein, which gives us more experimentally validated mutational consequence measurements than ever before. Such advances, together with ever larger sequence and structure databases, have created an opportunity to apply large scale analyses to coding variation, studying the effect on protein structure, function and phenotype. In this thesis I perform three large scale variant analyses. First, I use the consequences of variation to learn about protein structure and function. I compile a dataset from 28 deep mutational scanning studies, covering 6291 positions in 30 proteins, and use the consequences of mutation at each position to define a mutational landscape. I show rich biophysical relationships in this landscape and identify functionally distinct positional subtypes of each amino acid. In the second analysis, I explore genotype to phenotype prediction using a dataset of 1011 S. cerevisiae strains, with genotypes, transcriptomics, proteomics and measured phenotypes, and comprehensive gene deletions in four strains. I show knowledge-based models of mutational consequences and pathway function can be used to associate genes with phenotypes and predict growth phenotypes across 34 growth conditions. However, genetic background is found to have a large effect on variant consequences, to such an extent that the same deletion can be highly significant in one strain and have no effect in another. Finally, I analyse computational variant effect prediction, benchmarking current predictors using deep mutational scanning data. I then develop a new end-to-end deep convolutional neural network predictor that predicts consequences directly from sequence and structure and show it improves on current methods. Together these projects advance our knowledge of protein coding variation and enhance our capacity to link variation to impacts on structure, function and phenotype

Computational Approaches to Understanding the Structure, Dynamics, Functions, and Mechanisms of Various Bacterial Proteins

The 3D structure of a protein can be fundamentally useful for understanding protein function. In the absence of an experimentally determined structure, the most common way to obtain protein structures is to use homology modeling, or the mapping of the target sequence onto a closely related homolog with an available structure. However, despite recent efforts in structural biology, the 3D structures of many proteins remain unknown. Recent advances in genomic and metagenomic sequencing coupled with coevolution analysis and protein structure prediction have allowed for highly accurate models of proteins that were previously considered intractable to model due to the lack of suitable templates. Structural models obtained from homology modeling, coevolution-based modeling, or crystallography can then be used with other computational tools such as small molecule docking or molecular dynamics (MD) simulations to help understand protein function, dynamics, and mechanism.Here coevolution-based modeling was used to build a structural model of the HgcAB complex involved in mercury methylation (Chapter I). Based on the model it was proposed that conserved cysteines in HgcB are involved in shuttling mercury, methylmercury, or both. MD simulations and docking to a homology model of E. coli inosine monophosphate dehydrogenase (IMPDH) provided insights into how a single amino acid mutation could relieve inhibition by altering protein structure and dynamics (Chapter II). Coevolution-based structure prediction was also combined with docking, and experimental activity data to generate machine learning models that predict enzyme substrate scope for a series of bacterial nitrilases (Chapter III). Machine learning was also used to identify physicochemical properties that describe outer membrane permeability and efflux in E. coli and P. aeruginosa and new efflux pump inhibitors for the E. coli AcrAB-TolC efflux pump were identified using existing physicochemical guidelines in combination with small molecule docking to a homology model of AcrA (Chapter IV). Lastly, quantum mechanical/molecular mechanical simulations were used to study the mechanism of a key proton transfer step in Toho-1 beta-lactamase using experimentally determined structures of both the apo and cefotaxime-bound forms. These simulations revealed that substrate binding promotes catalysis by enhancing the favorability of this initial proton transfer step (Chapter V)

Blind protein structure prediction using accelerated free-energy simulations.

We report a key proof of principle of a new acceleration method [Modeling Employing Limited Data (MELD)] for predicting protein structures by molecular dynamics simulation. It shows that such Boltzmann-satisfying techniques are now sufficiently fast and accurate to predict native protein structures in a limited test within the Critical Assessment of Structure Prediction (CASP) community-wide blind competition

Deep learning extends de novo protein modelling coverage of genomes using iteratively predicted structural constraints

The inapplicability of amino acid covariation methods to small protein families has limited their use for structural annotation of whole genomes. Recently, deep learning has shown promise in allowing accurate residue-residue contact prediction even for shallow sequence alignments. Here we introduce DMPfold, which uses deep learning to predict inter-atomic distance bounds, the main chain hydrogen bond network, and torsion angles, which it uses to build models in an iterative fashion. DMPfold produces more accurate models than two popular methods for a test set of CASP12 domains, and works just as well for transmembrane proteins. Applied to all Pfam domains without known structures, confident models for 25% of these so-called dark families were produced in under a week on a small 200 core cluster. DMPfold provides models for 16% of human proteome UniProt entries without structures, generates accurate models with fewer than 100 sequences in some cases, and is freely available.Comment: JGG and SMK contributed equally to the wor