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.

PROJECT NARRATIVE. The goal of this proposal is to combine synergistic advances in genetic code expansion (GCE) and quantitative, high-throughput microfluidics to enable the facile site-specific incorporation of noncanonical amino acids (ncAAs) into proteins expressed across >1,000 microfluidic reaction chambers at once. The aims described here focus on the application of these techniques to study the mechanism of pyridoxal 5’-phosphate (PLP)-dependent enzymes, a ubiquitous class of enzymes with significant biological and biotechnological importance. More broadly, the expected outcome of this project is the development and demonstration of a powerful new platform for the dissection of protein function with unprecedented precision and scale enabled by the resource-efficient application of ncAAs.