Computational Investigations of Biomolecular Mechanisms in Genomic Replication, Repair and Transcription

High fidelity maintenance of the genome is imperative to ensuring stability and proliferation of cells. The genetic material (DNA) of a cell faces a constant barrage of metabolic and environmental assaults throughout the its lifetime, ultimately leading to DNA damage. Left unchecked, DNA damage can result in genomic instability, inviting a cascade of mutations that initiate cancer and other aging disorders. Thus, a large area of focus has been dedicated to understanding how DNA is damaged, repaired, expressed and replicated. At the heart of these processes lie complex macromolecular dynamics coupled with intricate protein-DNA interactions. Through advanced computational techniques it has become possible to probe these mechanisms at the atomic level, providing a physical basis to describe biomolecular phenomena. To this end, we have performed studies aimed at elucidating the dynamics and interactions intrinsic to the functionality of biomolecules critical to maintaining genomic integrity: modeling the DNA editing mechanism of DNA polymerase III, uncovering the DNA damage recognition/repair mechanism of thymine DNA glycosylase and linking genetic disease to the functional dynamics of the pre-initiation complex transcription machinery. Collectively, our results elucidate the dynamic interplay between proteins and DNA, further broadening our understanding of these complex processes involved with genomic maintenance

Nonergodic Jackson networks with infinite supply–local stabilization and local equilibrium analysis

Classical Jackson networks are a well-established tool for the analysis of complex systems. In this paper we analyze Jackson networks with the additional features that (i) nodes may have an infinite supply of low priority work and (ii) nodes may be unstable in the sense that the queue length at these nodes grows beyond any bound. We provide the limiting distribution of the queue length distribution at stable nodes, which turns out to be of product form. A key step in establishing this result is the development of a new algorithm based on adjusted traffic equations for detecting unstable nodes. Our results complement the results known in the literature for the subcases of Jackson networks with either infinite supply nodes or unstable nodes by providing an analysis of the significantly more challenging case of networks with both types of nonstandard node present. Building on our product-form results, we provide closed-form solutions for common customer and system oriented performance measures

Dynamics of protein interactions with new biomimetic interfaces: toward blood-compatible biomaterials

2019 Fall.Includes bibliographical references.Nonspecific blood protein adsorption on the surfaces is the first event that occurs within seconds when a biomaterial comes into contact with blood. This phenomenon may ultimately lead to significant adverse biological responses. Therefore, preventing blood protein adsorption on biomaterial surfaces is a prerequisite towards designing blood-compatible artificial surfaces. This project aims to address this problem by engineering surfaces that mimic the inside surface of blood vessels, which is the only known material that is completely blood-compatible. The inside surface of blood vessels presents a carbohydrate-rich, gel-like, dynamic surface layer called the endothelial glycocalyx. The polysaccharides in the glycocalyx include polyanionic glycosaminoglycans (GAGs). This polysaccharide-rich surface has excellent and unique blood compatibility. We developed a technique for preparing and characterizing dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. The glycocalyx-mimetic surfaces were prepared by adsorbing heparin- or chondroitin sulfate-containing polyelectrolyte complex nanoparticles (PCNs) to chitosan-hyaluronan polyelectrolyte multilayers (PEMs). We then studied in detail the interactions of two important blood proteins (albumin and fibrinogen) with these glycocalyx mimics. Surface plasmon resonance (SPR) is a common ensemble averaging technique for detection of biomolecular interactions. SPR was used to quantify the amount of protein adsorption on these surfaces. Moreover, single-molecule microscopy along with advanced particle tracking were used to directly study the interaction of single-molecule proteins with synthetic surfaces. Finally, we developed a groundwork for a kinetic model of long-term protein adsorption on biomaterial surfaces. In the first chapter, we thoroughly summarize the important blood-material interactions that regulate blood compatibility, organize recent developments in this field from a materials perspective, and recommend areas for future research. In the second chapter, we report the preparation and characterization of dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. In the third chapter, we investigate how combining surface plasmon resonance, X-ray spectroscopy, atomic force microscopy, and single-molecule total internal reflection fluorescence microscopy provides a more complete picture of protein adsorption on ultralow fouling polyelectrolyte multilayer and polymer brush surfaces, over different regimes of protein concentration. In the fourth chapter, the interactions of two important proteins from the blood (albumin and fibrinogen) with glycocalyx-mimetic surfaces are revealed in detail using surface plasmon resonance and single-molecule microscopy. Finally, in the fifth chapter, the long-term protein interactions with different biomaterial surfaces are studied with single-molecule microscopy an

Mass Spectrometry-Based Strategies in Protein Higher Order Structure Analysis: Fundamentals and Applications in Protein-Ligand Interactions

Protein ligand interaction is a fundamental question in biology and biochemistry, and many approaches including X-ray crystallography, nuclear magnetic resonance, cryogenic electron microscopy, mass spectroscopy (MS), infrared spectroscopy, circular dichroism, fluorescence spectroscopy and many others have been applied to address this question. Among these techniques, mass spectroscopy has the advantage of high throughput, low sample amount requirement, and mid-to-high spatial resolution. One of the MS-based approaches is protein footprinting, which utilizes labeling reagents to map the solvent accessible surface of the protein of interest thus deliver structural information. Irreversible labeling is represented by covalent labeling and radical labeling, in which labeling reagents react with amino acid side chains. Reversible labeling, on the other hand, is represented by hydrogen deuterium exchange (HDX), allowing the analysis of protein backbone. This dissertation describes the development of mass spectrometry-based approaches for protein higher order structure analysis, with an emphasize on the characterization of protein-ligand interaction analysis. The dissertation is divided into seven chapters, five of them describe original research. The first chapter introduces the mass spectrometry-based protein footprinting for protein higher order structure analysis, including historical overview, basic principles, major applications, and recent advancements.Chapter 2 and 3 describes a novel mass spectrometry-based method, LITPOMS, which combines ligand titration, fast photochemical oxidation of proteins (FPOP), and mass spectrometry measurements to assess protein ligand binding stoichiometry, binding sites, binding orders, affinities, and allosteric behaviors. The method was first demonstrated by melittin – holo-calmodulin binding, whose binding stoichiometry is 1:1 (Chapter 2). Chapter 3 describes an application of the LITPOMS in characterizing the calcium-calmodulin binding system. As a result, the calcium binding sites, binding orders, site-specific binding affinities, and most importantly the allosteric behavior of calmodulin upon binding with calcium was revealed via a single experiment. Chapter 4 highlights a mechanistic study of FPOP chemistry, through selective 18O labeling coupled with mass spectrometry analysis, and revealed the amino acid-specific oxygen uptake pathways in the FPOP platform and further highlighted the potential of tailoring the FPOP labeling condition to address different biological questions. Chapter 5 and 6 report application of HDX in addressing novel biological questions. Chapter 5 presents a thorough analysis of tetraspanin CD53 and CD81 with their binding partners and revealed the importance of tetraspanin open conformation in facilitating the interaction network. Chapter 6 describes an HDX study of the interaction between Class II lanthipeptide synthetase HalM2 with its partner HalA2-LP. Through a two-temperature HDX workflow, the confidence of binding site assignment increases significantly, especially for weak binding systems. The last chapter highlights the perspective and future work. These chapters combine to demonstrate the scope of developing and adopting mass spectrometry-based approaches to characterize protein-ligand interactions

Defining the mechanism behind the self-association of therapeutic monoclonal antibodies using mass spectrometric techniques

Protein aggregation is responsible for a vast array of life-threatening protein based diseases as well as being an economic hurdle in biopharmaceutical development and manufacturing. Monoclonal antibodies represent the fastest growing class of biotherapeutics, with 53 antibodies in late phase clinical trials as of late 2015. Antibodies serve as ideal therapeutics due to their exquisite specificity and favourable safety profile. However, further therapeutic antibody development is hamstringed by uncontrolled self-association and aggregation which can occur at all stages of biotherapeutic development. Therefore, there is an urgent need for methods to dissect the mechanisms that drive uncontrolled self-association and protein aggregation. This thesis presents techniques which were applied to address the identification of aggregated material of a therapeutically relevant monoclonal antibody, and to characterise the mechanism responsible for driving oligomerisation. A combination of mass spectrometric techniques were employed to visualise the oligomeric species. Ion mobility spectrometry coupled to nanoelectrospray ionisation mass spectrometry was utilised to identify the oligomeric species formed under native conditions and to define the oligomers in terms of their mass and collision cross-sectional area. To characterise the regions responsible for driving oligomer formation, chemical cross-linking was employed to capture the oligomeric species in solution which were then analysed using tandem mass spectrometry. The initial dimer interaction was modelled using distance restraints obtained from the chemical cross-linking results and a model proposed that explains the oligomerisation events, and how runaway polymerisation can occur at higher concentrations. Finally, a powerful in vivo assay in the E. coli periplasm was developed to differentiate between aggregation and non-aggregation-prone sequences using single chain variable fragments (scFv) of the antibodies studied. The results presented demonstrate the applicability of the assay to molecules relevant to the biopharmaceutical sector; as an upstream platform for the identification of aggregation-prone sequences, prior to antibody production and development. Overall, the work presented within this thesis describes techniques that can be successfully applied to define the mechanism that underpins the self-association of a therapeutically-relevant monoclonal antibody. Furthermore, the study presents a novel in vivo assay that can be used to identify aggregation-prone sequences, and to develop them further by mutagenesis, which could be useful in protein development in the biopharmaceutical sector

Elucidating the Structural Dynamics of Alpha-Synuclein by Structural Mass Spectrometry

Parkinson’s disease (PD) is characterised by the deposition of insoluble Lewy Bodies (LBs) in dopaminergic neurons in the brain. LBs are primarily composed of a-Synuclein (aS), a 140-residue, intrinsically disordered protein which can self-associate and undergo a transition from disordered monomers into ordered b-sheet rich amyloid fibril architectures. Characterising the structural properties of early intermediates in aS amyloid assembly is crucial towards elucidating amyloid assembly mechanisms. This thesis presents the development and application of structural mass spectrometry (MS) based techniques to study the structure and dynamics of N-terminally acetylated aS (aSNTA). Findings capture how the conformation of aS correlates with its amyloid propensity. The conformational ensemble of aS along with variants which decrease/abolish amyloid assembly is shown. Ion mobility MS shows that monomeric aSNTA exists as a conformational ensemble populating partially compact conformational families in equilibrium with extended conformational families and this thesis explores the effect of perturbing the aS conformational ensemble on its amyloid assembly kinetics. aS is negatively charged under physiological conditions and is known to bind divalent metal ions. Upon addition of Ca2+, Mn2+ or Zn2+, multiple binding events occur and the conformational ensemble of aSNTA is shifted to compact conformations and the rate of amyloid assembly is increased. Oligomeric species populated during aS amyloid assembly are considered toxic drivers of neurodegeneration in PD but are difficult to study due to their transient form and heterogeneity. In this thesis, hydrogen-deuterium exchange MS is used to characterise the role of the N-terminal region in trapped oligomer assembly. Overall, this thesis aims to shine light on the use of structural MS to investigate the structure and dynamics of transient, heterogeneous proteinaceous species. Evidence is provided for the accelerating role of aS compaction in amyloid propensity and provides a foundation for the further development of structural MS based methodologies

A proteomics investigation into the role of zDHHC23 and MROH6 in neuroblastoma

Neuroblastoma (NB) is the most common malignant solid tumour diagnosed in infants, accounting for ~15% of all childhood cancer-related deaths. Current patient risk stratification criteria are heavily reliant on the presence of a MYCN amplification, albeit only accounting for ~25% of patients. The inadequate prognostic risk stratification of patients results in children receiving either inefficient or excessive treatment with a myriad of severe lifelong side effects for survivors. Therefore, the identification and characterisation of novel biomarkers could not only identify new therapeutic targets but could also improve risk stratification and treatment planning. A comparative transcriptomic analysis of NB tumours (obtained from the chick embryo model) grown under normal oxygen tensions (normoxia, 21% O2) or hypoxia (1% O2), a model for aggressive NB tumours that correlates with poor patient prognosis, identified multiple significantly upregulated genes in aggressive (hypoxic) tumours specifically, with Zinc Finger DHHC-Type Palmitoyltransferase 23 (zDHHC23) and Maestro Heat Like Repeat Family Member 6 (MROH6) exhibiting the best correlation with poor prognosis. This thesis sought to validate these expressed gene products as potential biomarkers in NB. I also investigated the molecular function of these two proteins under normoxic and hypoxic conditions, supplementing the currently limited available knowledge. Commercially available antibodies for these two proteins were unsuccessful for use in either immunostaining, a procedure currently used as the ‘gold-standard’ of clinical biomarker screening, or for immunoblotting of endogenous protein, with all of the antibodies evaluated lacking specificity. Although targeted mass spectrometry assays were successfully developed, they lacked the sensitivity to detect endogenous proteins, likely due to low levels of protein expression. Therefore, I focused on the biochemical characterisation of these two proteins, cloning dual reporter HA-mCherry-protein and protein-mCherry-HA plasmids to facilitate immunoprecipitation of exogenously expressed protein and evaluation of sub-cellular localisation. I developed and optimised a HA-tag based immunoprecipitation protocol for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to allow identification of post-translational modifications (PTMs) and protein interaction networks. These experiments revealed extensive hypoxia-induced regulation of protein binding partners, with ~70% of the interactome (from a total of 262 and 253 co-immunoprecipitated proteins for zDHHC23 and MROH6 respectively) changing as a function of O2 tension. GOterm analysis of these interactomes suggests that zDHHC23 is a component of several potentially important malignancy pathways, including cytoskeletal reorganisation and adhesion. Label free quantification analysis of MROH6 identifies high stoichiometric binding to Breast Cancer Anti-oestrogen Resistance protein 1 (BCAR1), inferring potential roles in telomere maintenance and genetic stability. Additionally, PTM analysis identified one and three phosphorylation sites on MROH6 and zDHHC23 respectively, with zDHHC23 S252 predicted to be regulated by Cyclin dependent kinases. Finally, I developed, to my knowledge, the first reported click-chemistry based high-throughput LC-MS/MS pipeline for the unbiased identification of zDHHC23 palmitoylated substrates, concluding that the ‘palmitome’ is much more complex than currently understood and likely regulates localisation to membrane bound organelles and extracellular vesicles, as well as its established role in plasma membrane localisation. Overall, using LC-MS/MS approaches, I explore and discuss how zDHHC23 and MROH6 overexpression may contribute to aggressive NB development and poor patient prognosis