Browsing by Author "McCullagh, Martin, advisor"
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Item Open Access Allostery of the flavivirus NS3 helicase and bacterial IGPS studied with molecular dynamics simulations(Colorado State University. Libraries, 2020) Davidson, Russell Bruce, author; McCullagh, Martin, advisor; Bernstein, Elliot, committee member; Barisas, George, committee member; Geiss, Brian, committee memberAllostery is a biochemical phenomenon where the binding of a molecule at one site in a biological macromolecule (e.g. a protein) results in a perturbation of activity or function at another distinct active site in the macromolecule's structure. Allosteric mechanisms are seen throughout biology and play important functions during cell signaling, enzyme activation, and metabolism regulation as well as genome transcription and replication processes. Biochemical studies have identified allosteric effects for numerous proteins, yet our understanding of the molecular mechanisms underlying allostery is still lacking. Molecular-level insights obtained from all-atom molecular dynamics simulations can drive our understanding and further experimentation on the allosteric mechanisms at play in a protein. This dissertation reports three such studies of allostery using molecular dynamics simulations in conjunction with other methods. Specifically, the first chapter introduces allostery and how computational simulation of proteins can provide insight into the mechanisms of allosteric enzymes. The second and third chapters are foundational studies of the flavivirus non-structural 3 (NS3) helicase. This enzyme hydrolyzes nucleoside triphosphate molecules to power the translocation of the enzyme along single-stranded RNA as well as the unwinding of double-stranded RNA; both the hydrolysis and helicase functions (translocation and unwinding) have allosteric mechanisms where the hydrolysis active site's ligand affects the protein-RNA interactions and bound RNA enhances the hydrolysis activity. Specifically, a bound RNA oligomer is seen to affect the behavior and positioning of waters within the hydrolysis active site, which is hypothesized to originate, in part, from the RNA-dependent conformational states of the RNA-binding loop. Additionally, the substrate states of the NTP hydrolysis reaction cycle are seen to affect protein-RNA interactions, which is hypothesized to drive unidirectional translocation of the enzyme along the RNA polymer. Finally, chapter four introduces a novel method to study the biophysical coupling between two active sites in a protein. The short-ranged residue-residue interactions within the protein's three dimensional structure are used to identify paths that connect the two active sites. This method is used to highlight the paths and residue-residue interactions that are important to the allosteric enhancement observed for the Thermatoga maritima imidazole glycerol phosphate synthase (IGPS) protein. Results from this new quantitative analysis have provided novel insights into the allosteric paths of IGPS. For both the NS3 and IGPS proteins, results presented in this dissertation have highlighted structural regions that may be targeted for small-molecule inhibition or mutagenesis studies. Towards this end, the future studies of both allosteric proteins as well as broader impacts of the presented research are discussed in the final chapter.Item Open Access Determining driving forces for small molecule aggregation using computational and theoretical methods(Colorado State University. Libraries, 2022) Anderson, Jakob Edward, author; Rappé, Anthony, advisor; McCullagh, Martin, advisor; Kennan, Alan, committee member; Chen, Eugene, committee member; Shipman, Patrick, committee memberMolecular aggregation is largely dictated by noncovalent interactions and is a phenomenon found in a broad list of disciplines. Computational and theoretical methods, such as molecular dynamics simulations and Quantum Mechanical calculations, are well suited techniques to study the noncovalent association of various systems as they provide atomistic resolution and experimentally comparable results for the timescales on which association occurs. The studies found in this dissertation are introduced in the first chapter and are put in the context of using computational methods to study the noncovalent association and aggregation of small molecules. Chapters two, three, and four provide a foundation for the rational design of dipeptides for a given application. A wide range of potential applications for diphenylalanine (FF) have been proposed which would benefit from the development of design principles. Chapter two discusses the complexity of the noncovalent interactions at multiple stages in the FF self-assembly process. Specifically, we suggest the initial aggregation of FF is predominantly driven by electrostatics, and after a reorientation event, nanotube growth is suggested to be driven by solvent mediated forces. The results from this chapter use an array of generalized analyses enabling quantitative comparisons to future dipeptide studies. The impact of sidechain modification for either FF residue is studied in chapter three by considering valine-phenylalanine (VF) and phenylalanine-valine (FV). While the monomeric conformations are shown to sample the same states for these two dipeptides, the probabilities for state sampling as well as the water dynamics around the peptide bond are shown to differ. Chapter four connects chapters two and three by considering both the behavior of sequence dependence and dimerization of VF, FV, isoleucine-phenylalanine, and phenylalanine-isoleucine relative to that of FF. The modification of the C-terminus of FF to a smaller hydrophobic sidechain is hypothesized to enable tighter packing from this study. Additionally, N-terminus FF modification is hypothesized to increase the solvent mediated forces during dimerization in agreement with the results from chapter three. While not a completed study, chapter four provides a foundation for the continued development of design principles for FF-derivatives. A novel approach to computing the free energy of association from Quantum Mechanical calculations is then described in chapter five. Due to the treatment of low energy frequencies as harmonic and a lack of temperature dependence, calculations of the entropy of associating molecules is inaccurate. The rigid-rotor-Gaussian-oscillator approximation proposed addresses these issues by treating low lying modes with anharmonic Gaussian potentials and wave functions as well as adding a temperature dependence to the partitioning between vibrational and translational/rotational modes. This approximation significantly reduces the error in computing the entropy of associating molecules resulting in a more accurate calculation of the total free energy. The results from these studies as well as future studies based on the work in this dissertation are then summarized in the final chapter.Item Open Access Implicit solvation using the superposition approximation applied to many-atom solvents with static geometry and electrostatic dipole(Colorado State University. Libraries, 2020) Mattson, Max Atticus, author; Krummel, Amber T., advisor; McCullagh, Martin, advisor; Szamel, Grzegorz, committee member; Prieto, Amy, committee member; Krueger, David, committee memberLarge-scale molecular aggregation of organic molecules, such as perylene diimides, is a phenomenon that continues to generate interest in the field of solar light-harvesting. Functionalization of the molecules can lead to different aggregate structures which in turn alter the spectroscopic properties of the molecules. To improve the next generation of perylene diimide solar cells a detailed understanding of their aggregation is necessary. A critical aid in understanding the spectroscopic properties of large-scale aggregating systems is molecular simulation. Thus development of an efficient and accurate method for simulating large-scale aggregating systems at dilute concentrations is imperative. The Implicit Solvation Using the Superposition Approximation model (IS-SPA) was originally developed to efficiently model nonpolar solvent–solute interactions for chargeless solutes in TIP3P water, improving the efficiency of dilute molecular simulations by two orders of magnitude. In the work presented here, IS-SPA is developed for charged solutes in chloroform solvent. Chloroform is the first solvent model developed for IS-SPA that is composed of more than one Lennard-Jones potential. Solvent distribution and force histograms were measured from all-atom explicit-solvent molecular dynamics simulations, instead of using analytic functions, and tested for Lennard-Jones sphere solutes of various sizes. The level of detail employed in describing the 3-dimensional structure of chloroform is tested by approximating chloroform as an ellipsoid, spheroid, and sphere by using 3-, 2-, and 1-dimensional distribution and force histograms respectively. A perylene diimide derivative, lumogen orange, was studied for its unfamiliar aggregation mechanism in chloroform and tetrahydrofuran solvents via Fourier-transform infrared and 2dimensional infrared spectroscopies as well as all-atom explicit-solvent molecular dynamics simulations and quantum mechanical frequency calculations. Molecular simulations identified two categories of likely aggregate dimer structures: the expected -stack structure, and a less familiar edge-sharing structure where the most highly charged atoms of the perylene diimide core are strongly interacting. Quantum mechanical vibrational frequency calculations were performed for various likely dimer aggregate structures identified in molecular simulation and compared to experimental spectroscopic results. The experimental spectra of the aggregating system share qualities with the edge-sharing dimer frequency calculations however larger aggregate structures should be tested. A violanthrone derivative, violanthrone-79 (V-79), was studied for its differing aggregation mechanisms in chloroform and tetrahydrofuran solvents via Fourier-transform infrared and 2dimensional infrared spectroscopies as well as all-atom explicit-solvent molecular dynamics simulations and quantum mechanical frequency calculations. The -stacking aggregate structure of V-79 is supported by all methods used, however, the type of -stacking orientations are different between the two solvents. Chloroform supports parallel -stacked aggregates while tetrahydrofuran supports anti-parallel -stacked aggregates which show differing vibrational energy delocalization between the aggregated molecules. The publications in chapters 3 and 4 demonstrate the power of combining experimental spectroscopy and computational methods like molecular dynamics simulations and quantum mechanical frequency calculations, however, they also show how having larger simulations with multiple solute molecules are needed. This is why developing IS-SPA to be used for these simulations is necessary. Further developments to IS-SPA are discussed regarding the importance of various symmetries of chloroform and the subsequent dimensionalities of the histograms used to describe its distribution and Lennard-Jones force. Two methods for describing the Coulombic forces of chloroform solvation are discussed and tested on oppositely charged Lennard-Jones sphere solutes. The radially symmetric treatment fails to capture the Coulombic forces of the spherical solute system from all-atom explicit-solvent molecular dynamics simulations. A dipole polarization treatment is presented and tested for the charged spherical solute system which better captures the Coulombic forces measured from all-atom explicit-solvent molecular dynamics simulations. Additional considerations for the improvement of IS-SPA and the developments in this work are presented. The dipole polarization approximation outlined in chapter 5 assumes that each chloroform is a static dipole, allowing the dipole magnitude to fluctuate as well as polarize is a more physically rigorous approximation that will likely improve the accuracy of Coulombic forces in IS-SPA. A novel method, drawn from the knowledge gained studying chloroform, for the efficient modeling of new solvent types including flexible solvent molecules in IS-SPA is discussed.Item Open Access Modeling conformational heterogeneity in biomolecules(Colorado State University. Libraries, 2023) Klem, Heidi, author; Paton, Robert, advisor; McCullagh, Martin, advisor; Levinger, Nancy, committee member; Kennan, Alan, committee member; Geiss, Brian, committee memberRegulation of biocatalytic cascades is essential for biological processes but has yet to be exploited in real-world applications. Allostery is a prime example, where binding of an effector molecule alters function in a remote location of the same biomolecule. V-type allostery is especially fascinating, as the reaction rate can be either increased or decreased in response to effector binding. Determining how conformational changes affect the reaction rate is challenging due to the disparity of timescales between the underlying molecular processes. Experimental methods, such as X-ray crystallography, can help to capture large-scale conformational change. However, the resulting structures are not guaranteed to correspond to the biophysical state relevant to the research questions being addressed. Structural changes that occur during the chemical reaction are particularly elusive to this approach. To understand the connection between conformational change and catalytic consequence, a description of the reaction mechanism and relevant configurations is needed. Quantum mechanical (QM) methods can be used to propose enzyme reaction mechanisms by modeling femtosecond motions of forming and breaking bonds. Large-scale conformational changes take place over much longer timescales that cannot be simulated at the QM level, therefore requiring classical simulation techniques. This dissertation focuses on the challenges posed by conformational change in the field of computational biocatalysis. The first chapter examines the prevalence of conformational change in enzymes, its relationship to catalysis, and the difficulties it presents. The second chapter looks at the influence of active site structural features on reaction rates in the allosteric enzyme IGPS using QM approaches and energy decomposition schemes. The third chapter covers the development of methods that use molecular dynamics (MD) simulations to analyze relevant structural states from simulation data and identify long-range communication pathways in biomolecules. The fourth chapter presents a Python code, enzyASM, that automates the generation of QM-based truncated active site models and discusses ongoing developments that will aid reproducibility and standardization in this field of research. The fifth and final chapter summarizes the implications of this Thesis work in computational biocatalysis and envisions how remaining challenges can be addressed to maximize potential to solve real-world problems.Item Open Access Molecular dynamics simulations of peptide and protein systems(Colorado State University. Libraries, 2021) Weber, Ryan Nicholas, author; McCullagh, Martin, advisor; Szamel, Grzegorz, committee member; Finke, Richard, committee member; Wang, Qiang, committee memberMolecular systems composed of amino acids play an important role in biological systems and have numerous functions and applications due to their enormous chemical versatility. These systems are usually divided into peptides and proteins based on the number of amino acids that compose each molecule. Molecular dynamics simulations can provide molecular-level insights into the self-assembly of peptide systems and the function of protein systems where experimental methods fail. Peptides are utilized for their switchable and self-assembling properties for the engineering of novel biomaterials which are responsive to external stimuli. Often, peptides are paired with aromatic molecules to incorporate interesting optoelectronic properties into the material. Chapter 2 discusses a molecular dynamics simulation study on the self-assembling properties of the self-complimentary (RXDX)4 sequence paired with an unnatural coumarin amino acid for the design of a pH-switchable, optoelectronic, self-assembling biomaterial. Specifically, it is found that the hydrophobicity of the peptide sequence plays a significant role in the stability and pH-switchability of (RXDX)4 and coumarin-(RXDX)4 β-sheet fibers. Proteins are essential to all known life and participate in nearly every cellular process. There are many varieties of proteins with important diverse functions. Helicase proteins hydrolyze NTP to catalyze the translocation and unwinding of double-stranded nucleic acids such as RNA and DNA and play a critical and extensive role in viral replication. Nsp13 is a helicase protein that is an important component of the viral replication machinery of the severe acute respiratory syndrome coronavirus-2 and remains a promising target for antiviral drugs. Chapter 3 presents a molecular dynamics simulation study on the ATP-dependent translocation mechanism of the SARS-CoV-2 nsp13 helicase. Specifically, the results from the study suggest that nsp13 may translocate using an inchworm stepping mechanism and that the binding of ATP may cause the first step in the translocation cycle. Motifs Ia, IV, and V are identified as key motifs in the translocation mechanism of nsp13 and as potential targets for the development of antiviral drugs against SARS-CoV-2. Although molecular dynamics simulation is a powerful approach to investigate condensed phase molecular phenomenon such as protein folding, allostery, and self-assembly, molecular dynamics is limited in the size and length of simulations that can be performed. Implicit solvent simulation methods, such as Implicit Solvation using the Superposition Approximation (IS-SPA), were developed to address these issues in solvated systems. The goal of IS-SPA is to improve the efficiency of molecular dynamics simulations by removing the solvent from the system, but still include the effect of the solvent on the solute. Chapter 4 presents the development and optimization of an IS-SPA molecular dynamics code on a GPU using CUDA. Specifically, the performance of three different IS-SPA CUDA algorithms are compared. The future studies of the self-assembly of peptide systems for the design of biomaterials, the ATP-dependent translocation mechanism of the SARS-CoV-2 nsp13, and the optimization of the GPU-capable IS-SPA molecular dynamics code in CUDA are discussed in the final chapter.Item Open Access The effect of resins on the aggregation behavior of asphaltenes(Colorado State University. Libraries, 2020) Derakhshani Molayousefi, Mortaza, author; McCullagh, Martin, advisor; Szamel, Grzegorz, committee member; Van Orden, Alan, committee member; Ettema, Robert, committee memberMillions of barrels of crude oil are extracted on a daily basis. Crude oil has four main components separated by the SARA fractionation method.1 Asphaltenes are the heaviest component of the cured oil. They are known to be responsible for clogging oil wellbores and pipelines, which bedevils the oil industry financially. Additionally, the cleaning chemicals and the clogging waste has a huge negative impact on our environment. The majority of the research on understanding the clogging problem is focused on the asphaltenes as a fraction of crude oil without much consideration for the effects of specific chemical structure. Moreover, the role of other components of the crude oil such as resins is not clear. Here, we have performed structure specific studies of asphaltenes by performing all-atom molecular dynamics (MD) simulations to quantify the aggregation behavior of asphaltenes in the absence and presence of resins. In this research, we have studied the aggregation tendency of asphaltenes in connection with their molecular properties. Systems with 20 counts of model asphaltene molecules were studied for nanoaggregation behavior of eight model asphaltenes in their neat state. We have quantified the aggregation tendency of asphaltene molecules in n-heptane with isodesmic free energy of aggregation, ∆Giso, as well as a quantity called aggregation propensity (AP). Using ∆Giso and AP value, we have classified model asphaltene molecules to three main category of non-aggregating, mildly-aggregating, and readily-aggregating asphaltenes. Each category of asphaltene have different aggregation behavior. They differ in their molecular features that ultimately is related to their aggregation propensity. Subsequently, we have studied the aggregation tendency of asphaltene in the presence of resin with total of 48 systems comprising 8 model asphaltene molecules in the presence of 6 model resins. We wanted to determine the role of resins in the aggregation behavior of asphaltenes by observing the effect of presence of resin on the ∆Giso and AP values. Additional to ∆Giso, we have defined a normalized quantity called aggregation propensity ratio (APR) to compare the effect of resin on the aggregation of asphaltenes. Resins studied in this work had no promoting effect on the aggregation tendency of asphaltenes. In general, both ∆Giso and APR metrics suggest that aggregation of asphaltene in presence of resin is either not affected or is prevented to different degrees. We have studied the aggregation behavior of asphaltenes in nanoaggregation, clustering and flocculation stages proposed by Yen-Mullins model. Resins have from minimal disruptive to highly disruptive effect on the nanoaggregation of asphaltenes. We investigated the further aggregation of stable nanoaggregates into clustering and flocculation with 500 counts of mildly-aggregating and readily-aggregating asphaltene molecules. We found that both clustering and flocculation stages occur for the readily-aggregating asphaltenes and do not occur for the mildly-aggregating asphaltenes. Readily-aggregating asphaltene molecules with large negative ∆Giso and large AP values lead to clustering and flocculation whereas the mildly-aggregating asphaltenes stay in the form of nanoaggregates. Our results show that in order for asphaltenes to flocculate, there is a threshold for existence of adequate favorable molecular features. Asphaltenes containing large enough aromatic cores and/or heteroatom reach clustering and flocculation stages. Furthermore, we found that in the presence of a highly disruptive resin, clustering and flocculation does not occur. For the readily-aggregating asphaltenes the aggregation stops in the nanoaggregation stage and for the mildly-aggregating asphaltenes the size of the nanoaggregates decreases. Our results explain what kind of resins are capable of potentially solving the deposition problem with providing insight on the molecular features of both asphaltene and resin molecules. Such molecular insights paves the road to explore more natural based solutions in preventing the clogging problem in the oil industry by informed characterization of each oil reservoir and its capability to form aggregate or prevent aggregates within itself and in another reservoir.Item Open Access The flavivirus NS3 helicase Motif V controls unwinding function and alters viral pathogenesis in mosquitoes(Colorado State University. Libraries, 2020) Du Pont, Kelly Elizabeth, author; McCullagh, Martin, advisor; Geiss, Brian J., advisor; Szamel, Grzegorz, committee member; Snow, Christopher, committee member; Krummel, Amber, committee member; Ho, Shing, committee memberOver half of the world's population is at risk of flavivirus (e.g. dengue virus, West Nile virus, Japanese Encephalitis virus, and Zika virus) infection making it a global health concern. These specific mosquito-borne flaviviruses are responsible for causing a variety of symptoms and outcomes including flu-like fevers, encephalitis, hemorrhagic fevers, microcephaly, Guillain-Barré syndrome, and death. Unfortunately, vaccines and anti-viral therapeutics are not always effective in protecting against and treating viral infections. Sometimes these therapies cause more severe symptoms through an antibody dependent enhancement. Therefore, there is a pressing need for the development of effective anti-viral therapies against each flavivirus. For the advancement of these interventional strategies, a fundamental understanding of how flaviviruses replicate within hosts, including the mosquito vector, is required. This dissertation investigates how flaviviruses regulate viral replication, pathogenesis and mosquito transmission through the nonstructural protein 3 (NS3) helicase structure and function. A combination of virology, biochemistry, and computational simulations will be utilized to address how NS3 plays a role in viral infection, viral replication, and viral protein structure. An essential aspect of flaviviral genome replication is the unwinding of the double-stranded RNA intermediate via the C-terminal helicase domain of NS3. NS3 helicase translocates along and unwinds the double-stranded nucleic acids in an ATP-dependent manner. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well understood. Previous simulations in the group led us to hypothesize that Motif V is a critical component of the transduction mechanism. Here, we tested Motif V mutations in both sub-genomic replicon and recombinant protein systems to examine viral genome replication, helicase unwinding activity, ATP hydrolysis activity, and RNA binding affinity activity. NS3 helicase mutants, T407A and S411A, indicated reduced viral genome replication and increased turnover rates of helicase unwinding activity by a factor of 1.7 and 3.5 respectively. Additionally, we simulated Motif V mutants to probe the structural changes within NS3 helicase caused by the mutations. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. Motif V mutations T407A and S411A exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during viral genome replication. Next, we utilized T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo to probe the how these mutations play a role in pathogenesis and transmission of flaviviruses. Of the two Kunjin virus mutants, only S411A Kunjin virus was recovered. In cell culture, S411A Kunjin decreased viral infection and increased cytopathogenicity as compared to WT Kunjin. Similarly, in surviving Culex quinquefasciatus mosquitoes, S411A Kunjin decreased infection rates as compared to WT Kunjin, but S411A Kunjin infection increased mortality compared with that of WT Kunjin infection. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these computational simulation, biochemical assay, and virology data indicate that flavivirus NS3 helicase Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus, not just regulation of translocation and unwinding during viral genome replication. The molecular level insights presented in this dissertation provide the fundamental research for understanding how to target specific regions of NS3 helicase for the advancement of anti-viral therapeutics.