Presentation Title
Investigation of VRK1 Point Variants
Abstract
Vaccinia-related kinase 1 (VRK1) is a protein kinase that phosphorylates a variety of transcription factors and is associated with the regulation of cellular processing. The kinase activity of the VRK1 protein is largely controlled by the substitute folding of the C-terminal tail. Multiple point mutations in VRK1 are associated with degenerative neuromuscular disorders, including L195V, R89Q and Y213H. These mutations have been studied clinically in patients but have seldom been studied in vitro with purified proteins to determine changes in molecular activity and folding.
Y213 is located in the functionally important region of the kinase called the activation loop; it is hypothesized that the Y213H mutation may reduce kinase stability and/or substrate binding. R89 is located in the functionally important C-helix, and the R89Q mutation is hypothesized to cause an unstable C-terminal tail conformation in the inactive form of VRK1, resulting in VRK1 being active at inappropriate times during cellular division. L195 is located in the core of the protein near the active site, and the mutant L195V may facilitate interactions with ATP; this mutant has been previously associated with increased phosphorylation of VRK1, p53, and histone H3. In vitro experiments are needed to examine the molecular-level reasons why these mutations cause these known physiological effects.
VRK1 L195V, R89Q and Y213H mutations were made to plasmids containing a His-tagged construct of the VRK1 kinase domain. The point mutant proteins were purified and then analyzed by circular dichroism and thermal denaturation to determine their stability and nucleotide affinity. Protein modeling in PyMOL and other programs was used for the conceptual visualization of the kinase and its associated changes due to these mutations.
Faculty Mentor: Emily Ruff
College
College of Science & Engineering
Department
Chemistry
Breakout Room
11
Start Date
4-14-2021 2:00 PM
End Date
4-14-2021 2:45 PM
Presentation Type
Video (Live-Zoom)
Investigation of VRK1 Point Variants
Vaccinia-related kinase 1 (VRK1) is a protein kinase that phosphorylates a variety of transcription factors and is associated with the regulation of cellular processing. The kinase activity of the VRK1 protein is largely controlled by the substitute folding of the C-terminal tail. Multiple point mutations in VRK1 are associated with degenerative neuromuscular disorders, including L195V, R89Q and Y213H. These mutations have been studied clinically in patients but have seldom been studied in vitro with purified proteins to determine changes in molecular activity and folding.
Y213 is located in the functionally important region of the kinase called the activation loop; it is hypothesized that the Y213H mutation may reduce kinase stability and/or substrate binding. R89 is located in the functionally important C-helix, and the R89Q mutation is hypothesized to cause an unstable C-terminal tail conformation in the inactive form of VRK1, resulting in VRK1 being active at inappropriate times during cellular division. L195 is located in the core of the protein near the active site, and the mutant L195V may facilitate interactions with ATP; this mutant has been previously associated with increased phosphorylation of VRK1, p53, and histone H3. In vitro experiments are needed to examine the molecular-level reasons why these mutations cause these known physiological effects.
VRK1 L195V, R89Q and Y213H mutations were made to plasmids containing a His-tagged construct of the VRK1 kinase domain. The point mutant proteins were purified and then analyzed by circular dichroism and thermal denaturation to determine their stability and nucleotide affinity. Protein modeling in PyMOL and other programs was used for the conceptual visualization of the kinase and its associated changes due to these mutations.
Faculty Mentor: Emily Ruff
Comments
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