Theses and Dissertations at Montana State University (MSU)
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Item Biophysical characterization of P22 bacteriophage and adenoassociated viruses(Montana State University - Bozeman, College of Letters & Science, 2016) Kant, Ravi; Chairperson, Graduate Committee: Brian Bothner; Aida Llauro, Vamseedhar Rayaprolu, Shefah Qazi, Pedro J. de Pablo, Trevor Douglas and Brian Bothner were co-authors of the article, 'Stability, biomechanics and structural changes in P22 bacteriophage during maturation' which is contained within this thesis.; Vamseedhar Rayaprolu and Brian Bothner were co-authors of the article, 'Understanding of P22 bacteriophage maturation by QCM-D' which is contained within this thesis.; Navid Movahed, Dewey Brooke, Antonette Bennett, Mavis Agbandje-McKenna and Brian Bothner were co-authors of the article, 'Prolonged incubation with liposome leads to PLA2 activation in adeno-associated viruses' which is contained within this thesis.; Vamseedhar Rayaprolu and Brian Bothner were co-authors of the article, 'Comparison of the visco-elastic properties of viruses, virus based nanomaterials and active protein cages' which is contained within this thesis.The dsDNA tailed bacteriophages comprise the largest evolving life form in the biosphere. They are not only the most abundant organism on Earth, but also plausibly the most ancient. The ancient origin of phage suggests that they have had the ample opportunity to undergo the evolutionary changes necessary to perform intricate coordinated biological functions. Therefore, characterizing a tailed bacteriophage will help not only to understand biology, but also help us to establish a relationship between structure and function. Viruses display a dynamic equilibrium between structural conformations, stability, flexibility and rigidity which is essential for the perpetuation of life cycle. Understanding this complex biophysical relationship is a daunting task and requires a combination of multidimensional approaches. P22 is a tailed bacteriophage and displays a series of structural transitions during maturation. To understand the important biophysical changes in the P22 at different stages of maturation, we have introduced a suite of orthogonal techniques to address the distinct properties of intermediates. These include Differential Scanning Fluorimetry which probes the thermal stability of P22 capsids, Hydrogen-Deuterium Mass Spectrometry, which probes the conformational flexibility and Atomic Force Microscopy and Quartz Crystal Microbalance with dissipation, which probe the biomechanical transformation in the capsids. P22 investigation using these techniques reveals the large scale structural arrangements along with the expansion. Global rearrangement results in an increase in stability, rigidity and reduced dynamics. The sum results of these studies indicate that expansion is accompanied by large scale inter-subunit rearrangements which lead to the enhanced hydrophobic core at different quasi-equivalent axes. We have also studied Adeno-associated viruses, which is used as a gene delivery vehicle for the treatment of genetic disorders. AAVs lipase contains a lipase domain and its activation is important for the successful infection. Activation mechanism of lipase domain is not thoroughly understood. To understand the mechanism, we have developed a Liquid Chromatography-Mass Spectrometry assay sensitive enough to measure lipase products. This assay confirms that prolonged incubation of AAVs with liposome is able to activate the lipase domain without the involvement of receptors and co-receptors.Item The development of hybrid biomaterials using the virus-like particle (VLP) from bacteriophage P22(Montana State University - Bozeman, College of Letters & Science, 2016) Edwards, Ethan James; Chairperson, Graduate Committee: Trevor Douglas; Rajarshi Roychoudhury, Benjamin Schwarz, Paul Jordan, John Lisher and Trevor Douglas were co-authors of the article, 'Co-localization of catalysts within a protein cage leads to efficient photochemical NADH and/or hydrogen production' which is contained within this thesis.; Dissertation contains several articles of which Ethan James Edwards is not the main author.A broad range of bio-composite materials have been developed through inspiration from biology. In particular, natural systems that confine, co-localize and protect their contents has inspired the design and synthesis of the P22 virus-like particle (VLP) to effect a suite of biomaterials. These materials were realized by taking advantage of the native protein architecture of P22 as an initiation point and platform for material synthesis. Introducing a reactive cysteine on the P22 coat protein provided an initiation point for polymer synthesis. Atom transfer radical polymerization (ATRP) was initiated creating a polymer framework on the interior of the P22 VLP. Using this polymerization technique (ATRP) a photocatalytic crosslinker was successfully incorporated for reduction of methyl viologen. Next, a manganese porphyrin imaging agent was loaded creating a T 1-enhanced MRI contrast agent, as an alternative to the highly toxic Gadolinium currently used. Inspired by photosynthetic machinery, the P22-xAEMA system was labeled with a co-catalyst system, creating a co-localized photocatalytic nanoparticle capable of photochemically producing NADH/hydrogen. The production was controlled by labeling density of catalysts resulting in a tunable biomaterial. The design of a complex bio-hybrid material was developed by combining both synthetic and genetic approaches. Coupling the enzyme Alcohol Dehydrogenase D from Pyrococcus furiosis with a small molecule catalyst led to a coupled catalytic system between a synthetic catalyst and biologically derived enzyme. The P22 VLP system was studied by atomic force microscopy (AFM) and cryoelectron microscopy (cryo-EM) unraveling its biophysical properties and providing insights for further material design. 2D-crystal arrays were formed from a variety of P22-protein biomaterials, for the development of functional P22 arrays. Lastly, the P22 VLP was monitored by charge detection mass spectrometry, giving insight into the stability of the scaffolding protein. These studies show the versatility of this system for both material synthesis and fundamental biochemical understandings. Overall, the work here continues to progress and push the boundaries of protein cage nanoparticles as platforms for material synthesis. The development of hybrid biomaterials from VLPs serve to improve our basic understandings of the natural systems they are derived from and provide additional design principles for improved complex biohybrid materials.Item Expression and purification of two CRISPR-CAS proteins, Csm3 and Csm5 from Mycobacterium tuberculosis(Montana State University - Bozeman, College of Letters & Science, 2015) Hashimi, Marziah; Chairperson, Graduate Committee: C. Martin LawrenceOne third of the World's population is infected with tuberculosis (TB). TB disease is caused by bacterium called Mycobacterim tuberculosis, which is a facultative intracellular parasite that is transferred through the air from one person to another in close contact. A six month course of four antimicrobial drugs is the only current treatment for drug-sensitive TB. However, multi-drug resistance TB is difficult to treat. Phage therapy might be one answer as a treatment for multi-drug resistance TB. In order for phage therapy to have a chance against TB, the immune system of bacteria, known as CRISPR/Cas needs to be inhibited. Our lab has taken a structural and biochemical approach to try to understand the CRISPR/Cas system in M. tuberculosis. We have cloned, expressed, and purified individual Csm proteins from the H37Rv M. tuberculosis strain. Two Csm protein, Csm3 and Csm5 were successfully purified to homogeneity in yields suitable for structure and biochemical studies. While to date, each has failed to produce crystals, the ability to the express and purify each of these proteins will allow further biochemical characterization of Csm3 and Csm5.Item Engineering bacteriophage P22 as a nanomaterial(Montana State University - Bozeman, College of Letters & Science, 2013) O'Neil, Alison Linsley; Chairperson, Graduate Committee: Trevor Douglas; Courtney Reichhardt, Benjamin Johnson, Peter E. Prevelige and Trevor Douglas were co-authors of the article, 'Genetically programmed in vivo packaging and controlled release of protein cargo from bacteriophage P22' in the journal 'Angewandte chemie international edition' which is contained within this thesis.; Gautam Basu, Peter E. Prevelige and Trevor Douglas were co-authors of the article, 'Co-confinement of fluorescent proteins: spatially enforced communication of GFP and mCherry encapsulated within the P22 capsid' in the journal 'Biomacromolecules' which is contained within this thesis.; Peter E. Prevelige and Trevor Douglas were co-authors of the article, 'Encapsulation within the P22 capsid greatly improves the stability of a phosphotriesterase' submitted to the journal 'Advanced Functional Materials' which is contained within this thesis.The precise architectures of viruses and virus-like particles are highly advantageous in synthetic materials applications. These nano-size compartments are perfectly suited to act as containers of designed cargo. Not only can these nanocontainers be harnessed as active materials, but they can be exploited for examining the effects of in vivo "cell-like" crowding and confinement on the properties of the encapsulated cargo. The high concentration of many different types of mutually volume excluding macromolecules in the cell causes it to be a crowded and confining environment in which to carry out reactions. Herein, the molecular design of the bacteriophage P22 encapsulation system is described and utilized for the synthesis of active nanomaterials and to explore the effect of encapsulation on the entrapped proteins' properties. In the designed system, any gene can be inserted and results in the fusion of the insert to a truncated form of the P22 scaffold protein. This scaffold protein fusion templates the spontaneous in vivo assembly of P22 capsids and also acts as an encapsulation signal. Once encapsulated, we can examine how crowding and confinement affect inter-molecular communication and activity of the cargo molecules. The P22 system is unique in that the capsid morphology can be altered, without losing the encapsulated cargo, resulting in a doubling of the capsid volume. Thus, the encapsulated fusions can be examined at two different internal concentrations. The packaged capsids contain up to 300 copies of fusion and fill more than 24% of the internal volume with the internal concentration of the fusions in the millimolar range. Not only are these fusions densely and efficiently packaged, but they retain their activity. Described herein is the packaging of fluorescent proteins, enzymes, and active peptides. In all cases, it is shown that the enforced proximity via encapsulation greatly affects the fusions activity compared to the fusion free in solution. To expand the utility of the P22 capsid as a nanomaterial, the inherent asymmetry implored by the portal complex has also been exploited. The P22 encapsulation system has proved to be an effective and versatile vehicle for nanomaterials design.Item Monitoring protien cage nanopaticle morphology for applications in medicines and materials(Montana State University - Bozeman, College of Letters & Science, 2011) Johnson, Benjamin Lawrence; Chairperson, Graduate Committee: Trevor DouglasProtein cage nanoparticles are naturally occurring proteins found in all domains of life. The breadth of structural knowledge and the ability to modify protein cage nanoparticles both chemically and genetically set them apart for use as platforms for biomedical templates and materials synthesis. The work described herein focuses on the use of protein cage nanoparticles as a protective agent from a suite of viral pathogens. Protein cage nanoparticles exist in many different morphological forms both within a specific particle and between particles. It is essential to characterize these different states in order to engineer a protein cage nano particle for biomedical and materials synthesis. Described here is an expanded protocol for determining the morphological state with the bacteriophage P22 capsid. Using multiple techniques including multi angle light scattering, analytical ultra centrifugation, agarose gel electrophoresis and transmission electron microscopy these states are described and characterized. P22 exits in four different morphological states: the procapsid, empty shell, expanded shell and so-called "wiffleball". Also characterized in the work is the small heat shock protein from Methanococcus jannaschii, which exists in two morphological states. One of the states being the assembled 12 nm cage structure and the other state being a disassembled cage structure that is most commonly described at elevated temperatures. The characterization of these structures can aid in the understanding the mechanism of formation for the immunological phenomena induced bronchial associated lymphoid tissue.