Combined In Vitro and Computational Investigations of Protein Kinase Point Mutations for the Undergraduate Teaching Lab

Most of the >500 protein kinase domains encoded by the human genome function in signal transduction pathways controlling aspects of the cell life cycle. Errors in signaling due to overexpression or mutation of protein kinases can result in diseases such as cancer, and many disease‐linked mutations have been identified. A mutation's pathogenicity may stem from effects on such fundamental features as kinase stability, ligand binding, catalysis, and/or regulation. In this multi‐week project developed for first semester Biochemistry lab, students used computational tools and in vitro techniques to study single‐residue kinase mutations previously linked to disease. Instruction was done mainly through videos and written protocols to facilitate independent work. Outside the lab, students used scientific literature to develop hypotheses about a point mutation's in vitro effects on kinase stability and activity. They used PyMOL to add their point mutation to published active and inactive conformation structures of their kinase. They then refined and evaluated these structures using free online computational tools, and they investigated refined structures for conformational changes relative to wild‐type. In parallel wet lab experiments, students purified a His‐tagged bacterial expression construct of the same point variant kinase. They used Bradford assays and SDS PAGE to assess their purification. Finally, they used circular dichroism and thermal denaturation assays to determine whether their point mutation significantly changed kinase stability, secondary structure, and nucleotide binding. Students used their data to evaluate their hypotheses and propose further investigations in two written reports. Overall, students gained valuable computational and lab skills and experience in hypothesis‐driven research.