Polly Fordyce, PhD, Stanford University Webinar

Presentation & Panel Discussions

Moderator: Polly Fordyce, Ph.D., Assistant Professor of Genetics and Bioengineering and fellow of the ChEM-H Institute at Stanford University

Presenter: Craig Markin, Ph.D.

Presenter’s Bio: Craig Markin is a Canadian Institutes of Health Research (CIHR) 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 a variety of kinetic and thermodynamic constants for >1000 enzyme variants in parallel. Craig performed his graduate work with Prof. Leo Spyracopoulos at the University of Alberta, where he studied the molecular mechanisms of polyubiquitin chain synthesis and their subsequent recognition by partner proteins in the DNA damage response.

Abstract: Despite enormous advances in our 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 discover and explore using traditional, low-throughput assays. To address this, we 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, we measured Michaelis-Menten kinetics for multiple substrates and inhibition constants for ground and transition state analogs, comprising >5000 kinetic and thermodynamic constants. Interpreting these data using a framework we 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. Surprisingly, many mutations with compromised catalysis bind transition state analogs with equal or greater affinity, suggesting that efforts to design novel enzymes that accurately mimic transition state analog structures may not yield efficient catalysts.

We are currently using HT-MEK to study the functional consequences of natural sequence variation in a library of ~100 alkaline phosphatases, to identify allosteric sites in human protein tyrosine phosphatases, and determine the functional consequences of human disease-associated variants in these and other enzymes. Through this presentation and panel discussion, we hope to identify new opportunities to apply HT-MEK to address current needs in chemical biology.

Panelists: Fordyce’s Lab members, other guests (TBD), the foundation members