Dissecting enzyme function at scale using synergistic advances in microfluidics and genetic code expansion - PROJECT SUMMARY. Noncanonical amino acids (ncAAs) have myriad valuable applications in the biochemical and biophysical sciences. Their site-specific incorporation into proteins of interest can directly install systematically perturbed residues, sensitive biophysical probes, bio-orthogonal handles, and post-translational modifications (PTMs) at positions of interest. While promising, these applications have been greatly limited by costly materials and labor- intensive, low-yielding preparations. To realize the full potential of ncAAs, I will leverage the recently developed high-throughput microfluidic enzyme kinetics (HT-MEK) platform from the Fordyce and Herschlag laboratories at Stanford University to enable the parallel expression, purification, and quantitative assay of >1,000 ncAA- harboring protein variants on a single microfluidic device. With this approach, it will become feasible and routine to collect >10,000 gold-standard biochemical measurements of ncAA-containing proteins while using less material and effort than is typically required to collect a single such measurement. To illustrate the power and utility of this technique, I will first apply it towards understanding the catalytic mechanisms governing proton transfer at carbon in the model system alanine racemase (AlaR), an important pyridoxal 5’-phosphate (PLP)-dependent enzyme involved in cell-wall biosynthesis. PLP-dependent enzymes account for 4% of all classified enzymatic activities and ~1.5% of prokaryotic reading frames, and they are increasingly important in biotechnology. Although we have a reasonable understanding of how the small- molecule cofactor itself can influence catalysis, the specific contributions of the protein scaffold remain speculative, qualitative, or both. Previous studies that have used traditional site-directed mutagenesis—altering many properties simultaneously—and only examined a handful of variants have failed to deliver a unified view of how this enzyme achieves its catalytic proficiency. Here, I will use ncAAs on the HT-MEK device to systematically and precisely perturb the electrostatic properties of critical catalytic residues in the active site of AlaR—leaving other steric properties largely unaltered—across 96 different enzyme variants. Specifically, I will investigate how interactions in the active site act together to optimize this difficult proton transfer to: (1) be highly efficient at neutral pH; and (2) achieve an exquisite 106:1 regioselectivity among competing pathways for reprotonation of the reactive intermediate. The new training that I obtain from this project will greatly and uniquely expand my skillset at the interface of biocatalysis and mechanistic enzymology, leaving me poised to achieve my long-term goal of creating new enzymes to address enduring and emergent challenges in the biological and chemical sciences. More broadly, the development of reliable methods for the quantitative, high-throughput assay of hundreds of ncAA-harboring proteins is expected to have far-reaching impacts in all areas of biochemical and biophysical research with significant applied and therapeutic relevance.
