Theses and Dissertations at Montana State University (MSU)
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Item Biochemical and biophysical characterization of plastic degrading aromatic polyesterases(Montana State University - Bozeman, College of Letters & Science, 2019) Topuzlu, Ece; Chairperson, Graduate Committee: Valerie Copie; Brandon C. Knott and Mark D. Allen were authors and Japheth Gado, Harry P. Austin, Erika Erickson, Bryon S. Donohoe, Nicholas A. Rorrer, Fiona L. Kearns, Graham Dominick, Christopher W. Johnson, Valerie Copie, Christina M. Payne, H. Lee Woodcock, Gregg T. Beckham and John E. McGeehan were co-authors of the article, 'Structural and biochemical characterization of MHETASE' submitted to the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.As the world is producing more plastics than it can recycle, accumulation of manmade polymers in the environment is becoming one of the greatest global threats humanity is facing today. One of the major contributors to the plastics pollution problem is polyethylene terephthalate (PET), an aromatic polyester widely used in the packaging, beverage, garment and carpeting industries. As a response to the onslaught of plastics in the environment, fungi and bacteria are evolving metabolic pathways to convert plastics into useable energy sources. One of these organisms, a bacterium, Ideonella sakaiensis 201-F6, has recently been identified to convert PET into its monomers, terephthalic acid (TPA) and ethylene glycol (EG), and to use these compounds for energy and growth. I. sakaiensis' ability to convert PET is made possible by two enzymes, named PETase and MHETase. As a first step, PETase breaks down the insoluble substrate PET into a soluble major hydrolysis product - mono-(2- hydroxyethyl) terephthalate (MHET), which is then further hydrolyzed by MHETase into TPA and EG. Crystal structure of PETase, as well as some of its biochemical features, have been reported several times to date, but MHETase has remained largely uncharacterized. This work focuses on further discovery-driven biophysical and biochemical characterization of PETase, visualization of PETase activity on various polyester surfaces, as well as the structural and biochemical characterizations of the MHETase enzyme. We have found that several aspects of PETase-mediated substrate surface modification hydrolysis mechanisms differ depending on the specific mechanical and material characteristics of the substrate. We have also found that PETase is inhibited by BHET. Additionally, we have solved the crystal structure of MHETase. MHETase consists of an alpha/beta hydrolase domain, and a 'lid' domain, commonly seen in lipases. Molecular dynamics simulations revealed the mechanism of MHETase action. Through bioinformatics approaches, we have also identified mutants of interest for improved MHETase activity. Coincubation of MHETase with PETase affects PET turnover in a synergistic fashion. Taken together, this work provides additional insights into the mechanisms of action of the PETase and MHETase enzymes, which may open new avenue for bioremediation and removing plastics from the environment in a sustainable manner.Item Understanding the solution-phase biophysics and conformational dynamics of virus capsids(Montana State University - Bozeman, College of Letters & Science, 2013) Rayaprolu, Vamseedhar; Chairperson, Graduate Committee: Brian Bothner; Benjamin M. Manning, Trevor Douglas and Brian Bothner were co-authors of the article, 'Virus particles as active nanomaterials that can rapidly change their viscoelastic properties in response to dilute solutions' in the journal 'RSC softmatter' which is contained within this thesis.; Shannon Kruse, Navid Movahed, Tim Potter, Balasubramanian Venkatakrishnan, Bridget Lins, Antonette Bennett, Robert McKenna, Mavis Agbandje-McKenna and Brian Bothner were co-authors of the article, 'Comparative analysis of adeno associated virus capsid stability and dynamics' submitted to the journal 'Journal of virology' which is contained within this thesis.; Navid Movahed, Ravikant Chaudhary, Geoff Blatter, Alec Skuntz, Sue Brumfield, Jonathan K. Hilmer, Mark J. Young, Trevor Douglas and Brian Bothner were co-authors of the article, 'Learning new tricks from an old dog; studies of CCMV capsid swelling' submitted to the journal 'Journal of virology' which is contained within this thesis.Viruses are the most abundant form of life on the planet. Many forms are pathogenic and represent a major threat to human health, but viruses recently have been used as nanoscale tools for gene therapy, drug delivery and enzyme nanoreactors. Viruses have historically been viewed as static and rigid delivery vehicles, but over the last few decades they have been recognized as flexible structures. Their structural dynamics are a crucial element of their functionality. Characterizing the biophysical properties of these viruses is both challenging and exciting. We have developed and used a multidimensional approach to tackle this task. Our techniques include Differential Scanning Fluorimetry, which probes the melting temperatures of virus capsids by the use of a fluorescent dye and Hydrogen-Deuterium Mass Spectrometry, which investigates the flexibility of the virus capsid protein by following the change in mass when Hydrogen is exchanged with Deuterium. Flexible regions exchange more. The above techniques are well complemented by the use of size-exclusion chromatography, which differentiates virus capsids based on their hydrodynamic radius and Limited proteolysis which again probes dynamic regions of the capsids up to the amino acid level. We have studied two different systems, Cowpea chlorotic mottle virus (CCMV) and Adeno-associated virus (AAV) using these methodologies. The sum result of these assays indicate that, in case of CCMV, capsids can undergo structural transitions due to very subtle pH and cation concentrations and the capsid protein is capable of rigid body transitions which affect the stability, while maintaining most of the secondary structure. In the case of AAV, the inherent sequence differences explains only partially the differences in stability and proteolytic susceptibility.