Project Summary This project will provide data critical to advance our understanding of enzyme sequence/structure/function relationships. We will develop innovative microfluidic tools and techniques and apply them to systematically study enzymes at a high-throughput and quantitative level. In the prior granting period, we developed HT- MEK (High-Throughput Microfluidic Enzyme Kinetics), which enabled recombinant expression, purification, and deep functional characterization of >1500 enzymes in parallel and returns kinetic and thermodynamic constants (e.g. kcat/KM, kcat, KM, Ki) at unprecedented scale. Here, we extend this platform to provide a suite of techniques (HT-MES, High-Throughput Microfluidic Enzyme Stability) capable of quantifying kinetic and thermodynamic stabilities (e.g. ΔGfold, kunfold) with similar throughput. We will also enable site-specific incorporation of noncanonical amino acids to provide the capability to isolate and systematically the impact of particular physicochemical residue properties and to investigate the impacts of post-translational modifications. During the prior granting period, our application of HT-MEK to profile multiple substitutions at each position and multiple functional parameters for the model alkaline phosphatase PafA revealed that residues with similar functional effects on catalysis formed large and spatially contiguous ‘regions’ that extended from the active site to distal surfaces, and that different regions affected different aspects of function. Here, we will systematically apply HT-MEK/S to a variety of new systems to build upon these observations and develop and test new models of how enzymes attain their functions and their observed kinetic and thermodynamic constants. Specifically, we will map functional couplings between residues in PafA via double and multi-mutant cycles and test the degree to which observations from PafA generalize by applying HT-MEK/S to investigate other alkaline phosphatase superfamily members and to acyl phosphatases, which provide ideal enzymes for developing and testing predictive computational models. We will also profile impacts of all nonsynonymous single nucleotide substitutions on folding and catalysis for protein tyrosine phosphatases (PTPs), providing clinically-relevant information about potential health consequences of mutations that can direct development of mechanistically relevant therapies and therapeutics. Finally, we will extend the reach of HT-MEK/S via collaborative projects with the Keedy and Zalatan labs. In all cases, we seek to use high-throughput data to test computational predictions, provide much-needed ground truth data for use by others, and reveal previously unattainable insights into the functional and energetic inner workings of enzymes.