Engagement of intrinsic disordered proteins in protein–protein interaction

Proteins from the intrinsically disordered group (IDP) focus the attention of many researchers engaged in protein structure analysis. The main criteria used in their identification are lack of secondary structure and significant structural variability. This variability takes forms that cannot be identified in the X-ray technique. In the present study, different criteria were used to assess the status of IDP proteins and their fragments recognized as intrinsically disordered regions (IDRs). The status of the hydrophobic core in proteins identified as IDPs and in their complexes was assessed. The status of IDRs as components of the ordering structure resulting from the construction of the hydrophobic core was also assessed. The hydrophobic core is understood as a structure encompassing the entire molecule in the form of a centrally located high concentration of hydrophobicity and a shell with a gradually decreasing level of hydrophobicity until it reaches a level close to zero on the protein surface. It is a model assuming that the protein folding process follows a micellization pattern aiming at exposing polar residues on the surface, with the simultaneous isolation of hydrophobic amino acids from the polar aquatic environment. The use of the model of hydrophobicity distribution in proteins in the form of the 3D Gaussian distribution described on the protein particle introduces the possibility of assessing the degree of similarity to the assumed micelle-like distribution and also enables the identification of deviations and mismatch between the actual distribution and the idealized distribution. The FOD (fuzzy oil drop) model and its modified FOD-M version allow for the quantitative assessment of these differences and the assessment of the relationship of these areas to the protein function. In the present work, the sections of IDRs in protein complexes classified as IDPs are analyzed. The classification “disordered” in the structural sense (lack of secondary structure or high flexibility) does not always entail a mismatch with the structure of the hydrophobic core. Particularly, the interface area, often consisting of IDRs, in many analyzed complexes shows the compliance of the hydrophobicity distribution with the idealized distribution, which proves that matching to the structure of the hydrophobic core does not require secondary structure ordering

New Methods to Study Proline-Rich Disordered Regions and Their Structural Ensembles in Protein Signaling Pathways

Ph.DDOCTOR OF PHILOSOPH

Investigation of membrane protein dynamics of gamma-glutamyl carboxylase using liquid chromatography and mass spectrometry

Membrane proteins are involved in numerous biological processes, including transport, signal transduction, and a variety of metabolic pathways. Despite their abundance, however, membrane proteins largely remain resistant to biophysical characterization due to complexities in sample preparation and limited knowledge regarding structural elucidation. The overlying objective of the work reported in this dissertation is focused upon developing proteomics based approaches for the structural investigation of integral membrane proteins involved in the vitamin K cycle. Vitamin K is an essential micronutrient that functions as a coenzyme in the carboxylation of vitamin K-dependent (VKD) proteins by the integral membrane protein Gamma-Glutamyl Carboxylase (GGCX). Concomitant with VKD modification, vitamin K is regenerated by a mechanism involving the enzyme Vitamin K Epoxide Reductase (VKOR) and the cycle continues. Structural analysis of GGCX and VKOR is of particular interest in understanding their functional involvement in blood coagulation, calcification, and cell growth control. Although the mechanisms for carboxylation and epoxidation have been investigated for over thirty years, biological recognition involving structural conformations and protein associations are not yet completely understood. As an alternative approach to classical biochemical experimentation, methods were developed to investigate GGCX protein dynamics by ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry. Following a brief overview of the vitamin K cycle (Chapter 1), Chapters 2-4 aim to develop analytical approaches for identifying the catalytic active site in GGCX using covalent cross-linking mass spectrometry. A comprehensive bottom-up proteomics methodology (Chapter 2) was applied to identify site-specific covalent attachment of synthetically modified VKD cross-linker substrates in Chapters 3 and 4. In Chapter 5, a new class of model membrane, Nanodiscs, are introduced providing a controlled, native phospholipid structure in which membrane proteins can be isolated in a water-soluble environment. Incorporation of GGCX embedded Nanodiscs are further investigated by hydrogen exchange mass spectrometry (HX MS) in Chapter 6. This novel system demonstrates the first reported application for investigation of membrane protein dynamics in a near-native environment by HX MS. The work outlined in this dissertation not only offers significant advancements in the structural investigation of GGCX, but provides unique platforms in which to investigate other complex membrane protein systems

Computational Approaches To Anti-Toxin Therapies And Biomarker Identification

This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data. Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and several substrates. These experiments were combined with lead optimization techniques to create a potent irreversible inhibitor which protects 95% of cells in vitro. Dynamics studies on a TcdB cysteine protease domain were performed to an allosteric communication pathway. Comparative analysis of the static and dynamic properties of the TcdA and TcdB glucosyltransferase domains were carried out to determine the basis for the differential lethality of these toxins. Large scale biological data is readily available in the post-genomic era, but it can be difficult to effectively use that data. Two bioinformatics methods were developed to process whole-genome data. Software was developed to return all genes containing a motif in single genome. This provides a list of genes which may be within the same regulatory network or targeted by a specific DNA binding factor. A second bioinformatic method was created to link the data from genome-wide association studies (GWAS) to specific genes. GWAS studies are frequently subjected to statistical analysis, but mutations are rarely investigated structurally. HyDn-SNP-S allows a researcher to find mutations in a gene that correlate to a GWAS studied phenotype. Across human DNA polymerases, this resulted in strongly predictive haplotypes for breast and prostate cancer. Molecular dynamics applied to DNA Polymerase Lambda suggested a structural explanation for the decrease in polymerase fidelity with that mutant. When applied to Histone Deacetylases, mutations were found that alter substrate binding, and post-translational modification

Coarse-grained and atomistic modelling of phosphorylated intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are involved in many biological processes such as signalling, regulation and recognition. One of the main questions regarding IDPs is how sequence, structure and function are related. Phosphorylation, a type of post-translational modification prevalent in intrinsically disordered proteins and regions, is an example of how modifications at the sequence level can induce changes in structure and thereby influence function. The lack of well-defined tertiary structure in IDPs makes them better described by an ensemble of conformations than a single structure. Furthermore, it causes them to be more difficult to study than conventional proteins, so a combined approach of experimental and simulation techniques are often advantageous. However, simulations rely on appropriate models. In this thesis, the conformational ensembles of IDPs, especially the saliva protein statherin, have been investigated using both simulations with different models and the experimental techniques small-angle X-ray scattering and circular dichroism spectroscopy. The aims have been to contribute to the collection of available tools for studying IDPs, by investigating models, and to explore the link between sequence and structure of IDPs, with special focus on phosphorylation. It was shown that a coarse-grained "one bead per residue model" can be used to describe several different IDPs and provide an understanding of how protein length, charge distribution and salt concentration affects IDPs. Furthermore, by including a hydrophobic interaction the model could qualitatively describe the self-association of statherin and provide insight on the balance of interactions and entropy governing the process. The model was however shown to overestimate the compactness of longer and more phosphorylated IDPs. Turning to atomistic simulations, it was revealed that the conformational ensembles of phosphorylated IDPs are highly influenced by salt bridges forming between phosphorylated residues and arginine/lysine/C-terminus, such that over-stabilised salt bridges cause larger compaction than observed in experiments. Another force field could however detect phosphorylation-induced changes in global compaction and secondary structure and relate them to interactions between specific residues, illustrating the potential ability of simulations to provide insight into phosphorylation

92nd Annual Meeting of the Virginia Academy of Science: Proceedings

Full proceedings of the 92nd Annual Meeting of the Virginia Academy of Science, May 13-15, 2014, Virginia Commonwealth University, Richmond, Virgini

Sequence Determinants of the Individual and Collective Behaviour of Intrinsically Disordered Proteins

Intrinsically disordered proteins and protein regions (IDPs) represent around thirty percent of the eukaryotic proteome. IDPs do not fold into a set three dimensional structure, but instead exist in an ensemble of inter-converting states. Despite being disordered, IDPs are decidedly not random; well-defined - albeit transient - local and long-range interactions give rise to an ensemble with distinct statistical biases over many length-scales. Among a variety of cellular roles, IDPs drive and modulate the formation of phase separated intracellular condensates, non-stoichiometric assemblies of protein and nucleic acid that serve many functions. In this work, we have explored how the amino acid sequence of IDPs determines their conformational behaviour, and how sequence and single chain behaviour influence their collective behaviour in the context of phase separation. In part I, in a series of studies, we used simulation, theory, and statistical analysis coupled with a wide range of experimental approaches to uncover novel rules that further explore how primary sequence and local structure influence the global and local behaviour of disordered proteins, with direct implications for protein function and evolution. We found that amino acid sidechains counteract the intrinsic collapse of the peptide backbone, priming the backbone for interaction and providing a fully reconciliatory explanation for the mechanism of action associated with the denaturants urea and GdmCl. We discovered that proline can engender a conformational buffering effect in IDPs to counteract standard electrostatic effects, and that the patterning those proline residues can be a crucial determinant of the conformational ensemble. We developed a series of tools for analysing primary sequences on a proteome wide scale and used them to discover that different organisms can have substantially different average sequence properties. Finally, we determined that for the normally folded protein NTL9, the unfolded state under folding conditions is relatively expanded but has well defined native and non-native structural preferences. In part II, we identified a novel mode of phase separation in biology, and explored how this could be tuned through sequence design. We discovered that phase separated liquids can be many orders of magnitude more dilute than simple mean-field theories would predict, and developed an analytic framework to explain and understand this phenomenon. Finally, we designed, developed and implemented a novel lattice-based simulation engine (PIMMS) to provide sequence-specific insight into the determinants of conformational behaviour and phase separation. PIMMS allows us to accurately and rapidly generate sequence-specific conformational ensembles and run simulations of hundreds of polymers with the goal of allowing us to systematically elucidate the link between primary sequence of phase separation

Protein Structure

Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic