Vamseedhar Rayaprolu —BIO-PROTOCOL

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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

David Paul Medical Research Council
13 protocols

Gal Haimovich Molecular Genetics, Weizmann Institute of Science, Israel
42 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
Reviewer merit
Editor 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

Authors: Vamseedhar Rayaprolu, Jolene Ramsey, Joseph Che-Yen Wang and Suchetana Mukhopadhyay, date: 03/20/2018, view: 1541, 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 ...

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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: 5376, 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 ...

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Kathrin Schlicht

Universität Bielefeld

Dear Sir or Madam

We have troubles to reach a virus concentration of 0,1 to 0,24 mg/mL. So our question is how you determined the protein concentration.

Yours sincerely

Kathrin Schlicht

3/2/2016 6:16:03 AM Reply

Vamseedhar Rayaprolu

Cell Biology and Neuroscience, Montana State University

Hi,

Sorry for the delayed response. Please look at the original reference (I posted the link in the previous post). Also look at references 51-54 in that link. This method was developed by our collaborators in SF9 using a baculoviral expression system. Let me know if you need more information.

2016-03-05 11:16:06

Kathrin Schlicht

Universität Bielefeld

Hi Vamseedhar,

we produce our AAVs by calcium phosphate-based transfection of plasmid DNA into HEK cells. The viral titer we reach is bellow the titer you need to get a protein concentration that high even after concentration by a molecular cut off filter. So we can`t perform DSF. We really like to test this method. Maybe you know how many HEK cells were transfected.

Thank you for your quick response.

2016-03-02 23:42:39

Vamseedhar Rayaprolu

Cell Biology and Neuroscience, Montana State University

Hi Kathrin,

We determined concentrations using gel densitometry. You could also use UV 280 to determine the protein concentration but because our virus sample also had DNA, we preferred running the gel and doing the densitometry. You can increase your protein concentration using molecular a weight cut off filter though AAV samples tend to stick to the filters quite a bit. The samples were obtained from our collaborators. For more information on purification of the virus and checking integrity, please refer to http://jvi.asm.org/content/87/24/13150.full

I hope this helps.

2016-03-02 08:25:51

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: 3114, 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: 2978, 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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10 Protocols edited

Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

Authors: Philipp A.M. Schmidpeter and Crina M. Nimigean, date: 10/05/2018, view: 311, Q&A: 0

The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart ...

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Retroviral Capsid Core Stability Assay

Authors: Tyler Milston Renner, Kasandra Bélanger and Marc-André Langlois, date: 09/20/2018, view: 589, Q&A: 0

Structural stability of the capsid core is a critical parameter for the productive infection of a cell by a retrovirus. Compromised stability can lead to premature core disassembly, exposure of replication intermediates to cytosolic nucleic acid ...

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