Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Abstract

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

Probing ion channel functional architecture and domain recombination compatibility by massively parallel domain insertion profiling

“…We propose that class organization is a universal feature of ion channels that results from constraints on channel structure to satisfy folding, assembly, and interaction with trafficking partners while providing flexibility for allosteric regulation and conformational changes during channel opening and closing. Other studies proposed similar ideas of spatially contiguous protein regions linked to specific functions: protein sectors based on co-evolution across homologs 85 , 97 – 99 , mapping regions involved in folding stabilization vs. conformational flexibility by circular permutation profiling 96 , or revealing the functional architecture of enzymes from high-throughput enzyme variant kinetics 100 . Shared among these studies is the concept of a perturbation (naturally occurring or engineered mutations, insertions) probing the underlying biophysical properties that directly impact sequence-function relationships.…”

Key concepts in muscle regeneration: muscle “cellular ecology” integrates a gestalt of cellular cross-talk, motility, and activity to remodel structure and restore function

“…Physical activity likely influences the resolution of inflammation, where the timing is critical; myotube alignment during regeneration; and the role of the ECM and ECM-bound ligands in guiding cells during movement. Myoblast migration (Roveimiab et al 2020 ) and the mechanisms of axonal targeting to neuromuscular junctions through synchronized myogenesis and nerve–muscle connectivity during regeneration (Daneshvar et al 2020 ) can be closely scrutinized by microfluidics applications, now used for high-throughput screening for mutations (Markin et al 2021 ) and biological assays (Grant et al 2018 ).…”

uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

“…38 High-throughput biochemistry reveals biophysical insights on an unprecedented scale, but constructing variant libraries has become the new rate-limiting step. 5 The ability of uPIC-M to generate the required variant libraries rapidly and efficiently will expand HTB to the study of new protein targets, systems, and questions.…”

“…This advance is made possible by developments in programmable automated liquid handling that increase the scale of plate-based assays, 1,2 and recently, by a miniaturized microfluidic platform that allows parallel measurement of thousands of variants on one microscope slide. [3][4][5] For basic enzymology and biophysics studies, HTB approaches using mutational scanning libraries allow identification of the effects of all residues on folding, stability, binding and catalysis. To advance precision medicine, libraries comprised of human allelic variants 6 can be assayed for folding and function and "variants of uncertain significance" can be classified by their biophysical propensity to drive disease or respond to therapeutics.…”

“…As high-throughput biochemistry tools increase the throughput of quantitative protein measurements by 10 2 -10 3 -fold, [1][2][3][4][5] generating the requisite variant libraries has emerged as the new bottleneck. For HTB to provide measurements for rationally chosen protein variants, input libraries must be user-defined clonal mutant libraries in which individual mutants are sequence-validated and physically isolated from one another for downstream assays.…”