Suchetana Mukhopadhyay Biology, Indiana University, USA, USA,
1 protocol

Joseph Wang Department of Biology, Indiana University, USA, USA,
1 protocol

Jolene Ramsey Department of Biology, Indiana University, USA, USA,
1 protocol

Brian Bothner Department of Chemistry and Biochemistry, Montana State University, USA
2 protocols
Balaji Olety Amaranath University of Massachusetts medical School
2 protocols

Balasubramanian Venkatakrishnan Syngene International Limited
3 protocols

Daniela Boehm Gladstone Institute of Virology and Immunology
2 protocols

David Paul Medical Research Council
16 protocols

Editor
Vamseedhar Rayaprolu
  • Research scientist, Cell Biology and Neuroscience, Montana State University
Research focus
  • Biophysics
  • Structural virology with a focus on viral assembly, functionalization and biology
    Membrane biology focused on stoichiometry and function
  • 2 Author merit

Education

PhD in Biochemistry, Montana State University, 2013

Lab information

Voltage sensing phosphatases (VSP) bridge two fundamental cell signals, voltage and phosphatidylinositol phosphate (PIPs) concentrations, through voltage regulated enzyme catalysis. The voltage sensing domain (VSD) from VSP senses membrane voltage changes triggering the cytosolic phosphatase domain (PD) to dephosphorylate PIPs. VSP activity directly links voltage to PIP concentrations, both of which are key components in cell excitability, migration and immune response. Although, we understand what reactions are catalyzed by VSP, the underlying biophysics and the physiological role are still unclear. Hence, understanding the biophysics of VSPs will play a critical role in identifying the biological context. My post-doctoral work in Dr. Kohout’s lab has shown that VSP from Ciona intestinalis (Ci-VSP) dimerizes predominantly through the VSD and that Ci-VSP dimers can act in a dominant-negative fashion. This is a paradigm shift in the VSP field. Ci-VSP was functionally considered a monomer although that work was done only at a single, low concentration of protein. My work in the paper explored a range of Ci-VSP concentrations and proved that the dimerization of Ci-VSP is a concentration dependent process. I utilized two-electrode voltage clamp fluorometry, co-immunoprecipitation and FRET based measurements to test Ci-VSP’s dimerization capability and its in vivo phosphatase activity. The functional impact of dimerization in VSPs across species could ultimately reveal how they may be involved in voltage dependent PIP signaling in cells.
http://www.montana.edu/cbn/faculty-staff/kohout.html

Publications

2 Protocols published
Alphavirus Purification Using Low-speed Spin Centrifugation
Authors:  Vamseedhar Rayaprolu, Jolene Ramsey, Joseph Che-Yen Wang and Suchetana Mukhopadhyay, date: 03/20/2018, view: 2750, Q&A: 2
Chemical and sedimentation procedures are used to purify virus particles. While these approaches are successful for wild-type viruses, they are often not feasible for purifying mutant viruses with assembly defects. We combined two published methods ...
Fluorometric Estimation of Viral Thermal Stability
Authors:  Vamseedhar Rayaprolu, Shannon Kruse, Ravi Kant, Navid Movahed, Dewey Brooke and Brian Bothner, date: 08/05/2014, view: 6413, Q&A: 1
Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a ...
8 Protocols reviewed
In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
Authors:  Grant Schauer, Jeff Finkelstein and Mike O’Donnell, date: 09/20/2017, view: 4298, Q&A: 0
The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, ...
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Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity
Authors:  Trung M. Hoang, Tatiana Kent and Richard T. Pomerantz, date: 06/20/2017, view: 3984, Q&A: 0
DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity ...
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14 Protocols edited
Viral Chromosome Conformation Capture (V3C) Assays for Identifying Trans-interaction Sites between Lytic Viruses and the Cellular Genome
Authors:  Kinjal Majumder, Maria Boftsi and David J Pintel, date: 03/20/2019, view: 467, Q&A: 0
The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding ...
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On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates
Authors:  Roland Remenyi, Raymond Li and Mark Harris, date: 02/20/2019, view: 864, Q&A: 0
Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of ...
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