Abstract
Voltage sensing phosphatases (VSPs) are one-of-a-kind proteins. They are regulated by voltage through a voltage sensing domain (VSD) similar to those in voltage-gated ion channels. Instead of a pore, however, the VSD is coupled to a phosphatidylinositol phosphate (PIP) phosphatase. As a result, VSPs are the only known protein with a direct link between voltage and phosphatase activity. VSPs form concentration-dependent dimers through interactions between their VSDs and phosphatase domains, yet the functional relevance of dimerization is not well understood. Both 3- and 5-phosphatase activity are observed at dimer-forming protein concentrations. When the concentration of VSP is significantly lowered, monomers and 5-phosphatase activity are detected. Furthermore, we observe only 5-phosphatase activity for dimers between functional and catalytically dead VSPs, suggesting 3-phosphatase activity requires two functional subunits. To confirm these changes to the substrate specificity, we are introducing mutations to monomerize VSP. The resulting proteins will be tested for dimerization with a coimmunoprecipitation and a single molecule pulldown assay (SiMPull). Preliminary results suggest multiple mutations are required to break the VSP dimer. We will also test functional differences between monomer and dimer VSPs by characterizing VSD motions using voltage clamp fluorometry and activity using FRET-based PIP biosensors.
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