Browsing by Author "Rayaprolu, Vamseedhar"

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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, Brian
The 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.
Curating viscoelastic properties of icosahedral viruses, virus-based nanomaterials, and protein cages
(2018-06) Kant, Ravi; Rayaprolu, Vamseedhar; McDonald, Kaitlyn; Bothner, Brian
The 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.
Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity
(2018-05) Rayaprolu, Vamseedhar; Royal, Perrine; Stengel, Karen; Sandoz, Guillaume; Kohout, Susy C.
Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P3 or PI(3,4)P2 Our results indicate that dimerization plays a significant role in Ci-VSP function.
Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase
(Rockefeller University Press, 2024-05) Rayaprolu, Vamseedhar; Miettinen, Heini M.; Baker, William D.; Young, Victoria C.; Fisher, Matthew; Mueller, Gwendolyn; Rankin, William O.; Kelley, John T.; Ratzan, William J.; Leong, Lee Min; Davisson, Joshua A.; Baker, Bradley J.; Kohout, Susy C.
The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1–S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme’s conformational response to membrane potential transients and influencing the function of the VSD.
Measuring Virus Rigidity by QCM-D
(2013-03) Ruzicka, Jesse; Bothner, Brian; Rayaprolu, Vamseedhar
The ability to study conformational changes of a virus can give us a glimpse of its intriguing structure, its infection cycle and more importantly its interactions with the host which enable it to cause the infection. These biophysical changes occur in-vivo due to different chemical environments and are of great interest. The effects of chemical changes such as; different pHs and salt concentrations can be studied in-vitro and can help us expand our knowledge about such virus particles. Many instruments and techniques are available for measuring such values. One instrument is the Quartz Crystal Microbalance with Dissipation Monitoring. The Q-SenseTM D300 instrument is capable of measuring both the fundamental frequency and the damping (dissipation) of the stored resonance energy of an excited quartz crystal. The measurement of these values allows us to calculate the rigidity of virus particles. Flock House Virus (FHV) and Nudaurelia Capensis Omega Virus (NѡV) were studied so that values of rigidity and insight to the virus’ viscoelastic properties could be obtained.
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.
The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells
(2019-04) Ratzan, Wil; Rayaprolu, Vamseedhar; Killian, Scott E.; Bradley, Roger S.; Kohout, Susy C.
Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.