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Coauthors
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
Coreviewers
Balaji Olety Amaranath University of Massachusetts medical School
2 protocols

Balasubramanian Venkatakrishnan Syngene International Limited
2 protocols

David Paul Medical Research Council
10 protocols

Gal Haimovich Molecular Genetics, Weizmann Institute of Science, Israel
41 protocols

Editor
Vamseedhar Rayaprolu
  • Research scientist, Cell Biology and Neuroscience, Montana State University
Research focus
  • Biophysics
  • Signaling cascades are the basis for cell life. From single cells to multi-cell organisms, cells must respond to and communicate with their local environment. The higher the organism the more complicated that communication becomes with interconnected and interdependent signaling pathways. Failures in these communication pathways often lead to diseases when the cells can no longer compensate.
    phosphatidylinositol signalingOne of the most important sites of cell signaling is the plasma membrane, where signals from the external environment are detected, physiological and biochemical signals of the cell initiated and through which ions, water, metabolites and proteins are selectively transported. These processes involve dozens of signaling pathways, and are critical in diverse cellular processes, including setting up and discharging the membrane potential, transforming cell morphology and migration, driving cell division and differentiation. One of the key links to the regulation of these diverse phenomena is phosphatidylinositol signaling (Fig. 1). Classically, control of this pathway has been attributed to soluble kinases and phosphatases that inter-convert phosphatidylinositol phosphates (PIPs) between forms that bind to distinct effectors. Since these effectors include ion channels and transporters, some of the fastest effects of PIP signaling are changes in membrane potential. Our research focuses on a recently discovered membrane protein that operates in the reverse direction: responding to changes in voltage to alter PIP levels. This protein, a voltage sensing phosphatase (VSP) that is found in diverse tissues including the nervous system, provides a fascinating feedback loop whose properties and physiological outcomes have yet to be determined.

    VSP belongs to a specialized family of voltage sensitive proteins that allow cells to transduce changes in membrane potential into chemical signals. Basic cellular processes, such as neuronal firing and muscle contraction, rely on these voltage dependent events. Voltage sensitive proteins respond to the changes in membrane potential via a voltage-sensing domain (VSD). The most common members of this family, the voltage-gated ion channels, use the VSD to open and close a pore domain (Fig. 2, left), which allows for the passive movement of ions down their concentration gradients. Instead of a pore, VSP has a phosphatase domain (Fig. 2, right) that dephosphorylates phosphates from PIP lipids. The phosphatase domain from VSP is quite similar to the tumor suppressor, PTEN, sharing 44% identity between the two catalytic domains.

    There are many challenges to understanding how membrane proteins function in the context of their native lipid environments. To probe this class of proteins, we apply voltage clamp fluorometry (VCF). VCF combines classical electrophysiological techniques with optical imaging to create a powerful technique to study full length membrane proteins in the context of a native cellular environment. By monitoring these protein motions in real time, the protein conformations can then be correlated with protein function giving a picture of how the protein moves to achieve its function.
  • 2 Author merit

Education

PhD in Biochemistry, Montana State University, 2013

Lab information

Kohout lab
http://www.montana.edu/cbn/faculty-staff/kohout.html

Publications

https://scholar.google.com/citations?user=oXymu_QAAAAJ&hl=en

2 Protocols published
Alphavirus Purification Using Low-speed Spin Centrifugation
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: 4794, 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: 2389, 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: 2321, 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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4 Protocols edited
BMV Propagation, Extraction and Purification Using Chromatographic Methods
Brome mosaic virus (BMV) is a well-known plant virus representing single-stranded RNA (ssRNA) positive-sense viruses. It has been widely used as a model in multiple studies concerning plant virus biology, epidemiology and the application of viral ...
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Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies
Authors:  Chie Aoki-Utsubo, Ming Chen and Hak Hotta, date: 05/20/2018, view: 780, Q&A: 0
In a narrow definition, virucidal activity represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to functionally inhibit (neutralize) viral infectivity ...
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