UCSF

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics: a QBI online seminar with Craig Markin

Date
Wednesday, February 24, 2021 - 11:00 am to 12:00 pm
Audience
students, staff, faculty, alumni, local science community
Location

Craig Markin is a postdoctoral fellow working jointly with Professors Dan Herschlag and Polly Fordyce at Stanford University, where he has developed HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a novel microfluidics-based assay to express, purify, and quantitatively measure libraries of >1000 enzyme variants across a battery of functional parameters. Craig performed his graduate work with Prof. Leo Spyracopoulos at the University of Alberta, studying the molecular mechanisms of polyubiquitin chain synthesis and their subsequent recognition by partner proteins in the DNA damage response. Craig is the recipient of a number of awards, including a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship.

Despite enormous advances in the understanding of enzyme function, how these catalysts achieve their rate enhancements and exquisite specificities remains elusive. Enzymes are highly cooperative, with interconnected residues and long-range interactions impossible to explore using traditional, low-throughput assays. To address this, Craig and his collaborators developed HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a platform to express, purify, and quantitatively measure libraries of variants across a battery of functional parameters. For >1,000 mutants of PafA, a highly proficient phosphatase, they measured Michaelis-Menten kinetics for multiple substrates, and inhibition constants for ground and transition state analogs, comprising a set of >5000 kinetic and thermodynamic constants. Interpreting these data using a framework they term Functional Component Analysis (FCA) revealed specific ‘regions’ of residues contributing to substrate specificity, ground state destabilization, and preferential transition state stabilization. These extended from the active site to the surface, potentially providing allosteric handles for rational manipulation of function. They anticipate that HT-MEK will enable approaches for identifying allosteric sites, help determine functional consequences of human disease-associated variants, and facilitate engineering of novel catalysts.

Talk title Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics
Hosts Tanja Kortemme and Tina Perica
Webinar ID 970 4843 2853
Passcode 411221

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