Clinton Taylor Email and Phone Number

Continuation of postdoctoral research activities with greater independence.

SPAK/OSR1 Kinase Protein-Protein Interaction Prediction and Validation- Conceived project, designed experiments, jointly led a team to acquire data, and performed all data analysis and interpretation. The majority of the techniques and methods had not previously been used by our research group.- Developed experimental methodology to probe protein-protein interaction specificity by utilizing peptide arrays, biophysical binding data, and affinity purification mass spectrometry.- Utilized a scoring algorithm to rank all instances of a known consensus binding motif. Filtered out false-positives and provided additional scoring based on conservation, cellular location, and solvent accessibility. Generated homology models of proteins for the solvent accessibility analyses.- Validated specific interaction predictions through biochemical and biophysical methods (co-IP, in vitro pull-downs, kinase assays) and through collaboration with other research groups.- Mentored an undergraduate student.OSR1 Kinase Regulation of Inward Rectifier K+ Channels Kir2.1 and Kir2.3- Based on results from the protein-protein interaction prediction project began a collaboration with another research group to study OSR1-mediated regulation of Kir2.1 and Kir2.3.- Designed experiments, jointly led a collaborative team to acquire data, analyzed data, interpreted results, and wrote manuscript that was accepted for publication.- Utilized biophysical techniques and molecular modeling to both confirm the interaction between OSR1 and Kir2.3 and to validate a novel alternate binding motif.- Determined that Kir2.1 and Kir2.3 are not direct substrates of OSR1 using both in vitro kinase assays and mass spectrometry analysis of post-translational modifications in HeLa cells.- Through collaboration, determined the mechanism of regulation by patch-clamp in HEK cells, and immunofluorescence combined with knockdown and pharmacological inhibition in HeLa cells.- Mentored a high school student.

MAP Kinase ERK2 E322K Cancer Mutational Hotspot Analysis- Designed and carried out experiments, analyzed and interpreted results, jointly directed two lab technicians, and jointly wrote manuscript.- Determined the crystal structures of ERK2 E322K and ERK2 D321N. Performed structural analysis to guide experiments and define the mechanism for E322K cancer enrichment.- Performed in vitro enzymatic assays to assess kinase activity and phosphatase activity in a variety of contexts. Employed plate-based (absorbance and fluorescence), 32P incorporation, and Western blot assays.- Biophysically characterized protein stability and signaling partner interactions using microscale thermophoresis, fluorescence polarization, and differential scanning fluorimetry.- Biochemically characterized signaling partner interactions by pull-downs of purified proteins and from lysates. Utilized affinity purification mass spectrometry to assess interactions in HeLa cells.SPAK Kinase Protein-Peptide Interaction Inhibitor High-Throughput Drug Screen- Conceived of project, developed methodology, and carried out experiments and analyses.- Developed fluorescence polarization-based assay suitable for high-throughput screening to find inhibitors of a protein-peptide interaction. Achieved assay Z-score greater than 0.9.- Screened a library of approximately 16,000 synthetic and natural product compounds.Structural and Functional Analysis of SPAK Kinase Activation Loop Domain Swap- Carried out experiments, analyzed and interpreted results, jointly wrote manuscript.- Determined crystal structure of SPAK kinase and performed structural analysis.- Carried out in vitro enzymatic assays to assess kinase activity (32P incorporation).- Utilized size-exclusion chromatography to assess kinase dimerization in multiple contexts.

Optimization of Expression and Purification of the CLOCK:BMAL1 Transcriptional Complex- Utilized bacterial and insect (Sf9) expression systems in combination with construct optimization to produce stable, high purity protein in quantities suitable for crystallization.- Worked as part of a team to carry out crystallization and structure solution.- This work resulted in the publication of a landmark paper in the field of circadian rhythms.

Lab Setup and Personnel Training- Worked as part of a small team to setup lab equipment and begin initial lab experiments.- Trained two incoming graduate students on common lab protocols and equipment use.Modular Polyketide Synthase Ketoreductases as Biocatalysts- Developed a method to analyze the stereospecificity of isolated ketoreductase domains through use of a coupled enzyme assay and HPLC chiral chromatographic separation.- As part of a team, determined crystal structures of multiple ketoreductase domains.- Performed chemical synthesis and purification of enzyme substrates.