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Item Curating viscoelastic properties of icosahedral viruses, virus-based nanomaterials, and protein cages(2018-06) Kant, Ravi; Rayaprolu, Vamseedhar; McDonald, Kaitlyn; Bothner, BrianThe beauty, symmetry, and functionality of icosahedral virus capsids has attracted the attention of biologists, physicists, and mathematicians ever since they were first observed. Viruses and protein cages assemble into functional architectures in a range of sizes, shapes, and symmetries. To fulfill their biological roles, these structures must self-assemble, resist stress, and are often dynamic. The increasing use of icosahedral capsids and cages in materials science has driven the need to quantify them in terms of structural properties such as rigidity, stiffness, and viscoelasticity. In this study, we employed Quartz Crystal Microbalance with Dissipation technology (QCM-D) to characterize and compare the mechanical rigidity of different protein cages and viruses. We attempted to unveil the relationships between rigidity, radius, shell thickness, and triangulation number. We show that the rigidity and triangulation numbers are inversely related to each other and the comparison of rigidity and radius also follows the same trend. Our results suggest that subunit orientation, protein–protein interactions, and protein–nucleic acid interactions are important for the resistance to deformation of these complexes, however, the relationships are complex and need to be explored further. The QCM-D based viscoelastic measurements presented here help us elucidate these relationships and show the future prospect of this technique in the field of physical virology and nano-biotechnology.Item Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation(2018-06) Kant, Ravi; Llauro, Aida; Rayaprolu, Vamseedhar; Qazi, Shefah; de Pablo, Pedro J.; Douglas, Trevor; Bothner, BrianThe capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.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 Decrease in pH destabilizes individual vault nanocages by weakening the inter-protein lateral interaction(2016-10) Llauro, Aida; Guerra, Pablo; Kant, Ravi; Bothner, Brian; Verdaguer, Nuria; de Pablo, Pedro JVault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time. Our experiments show that decreasing the pH of the solution destabilize the barrel region, the central part of vault particles, and leads to the aggregation of the cages. Additional analyses using Quartz-Crystal Microbalance (QCM) and Differential Scanning Fluorimetry (DSF) are consistent with our single molecule AFM experiments. The observed topographical defects suggest that low pH weakens the bonds between adjacent proteins. We hypothesize that the observed effects are related to the strong polar character of the protein-protein lateral interactions. Overall, our study unveils the mechanism for the influence of a biologically relevant range of pHs on the stability and dynamics of vault particles.