Dr Craig Markin

Division of Molecular and Cellular Function

Faculty of Biology Medicine and Health &

Manchester Institute of Biotechnology

University of Manchester

Abstract

A central challenge in biotechnology is the ability to efficiently and predictably harness the extraordinary catalytic power and specificity of natural enzymes for new reactions. While substrate binding and catalysis occur within enzyme active sites, these functions are frequently governed by extensive, energetically coupled networks of residues distributed throughout the entire protein structure (1). Such networks are largely invisible from sequence and structural information alone, and their identification requires quantitative measurements of the energetic consequences of mutations across whole enzymes.

HT-MEK (High-Throughput Microfluidic Enzyme Kinetics) is an emerging platform that enables quantitative enzymology at this scale (1,2). By integrating in situ protein expression, purification, and kinetic characterization, HT-MEK can measure up to 1,500 enzyme variants on a single microfluidic chip. The platform provides quantitative measurements of key functional parameters, including kcat, KM, and kcat/KM for multiple substrates, as well as inhibitor binding and protein stability (1,2).

I will discuss how HT-MEK is transforming our understanding of enzyme function, providing unprecedented insight into the cooperative residue networks that underpin the activity of highly evolved natural enzymes. Beyond fundamental discovery, I will explore how these quantitative sequence-function datasets can be leveraged to advance biocatalysis and accelerate the rational and machine learning-guided design of enzymes with improved or entirely new functions.

1. Markin CJ, Mokhtari DA, Sunden, F, Appel MJ, Akiva E, Longwell, SA, Sabatti C, Herschlag D, and Fordyce PM (2021) Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics. Science, 373, eabf8761

2. Markin CJ, Mokhtari DA, Du S, Doukov T, Sunden F, Cook JA, Fordyce PM, Herschlag D (2023) Decoupling of catalysis and transition state analog binding from mutations throughout a phosphatase revealed by high-throughput enzymology. Proc. Natl. Acad. Sci. U.S.A, 120, e2219074120

Biography

Craig completed his graduate training in the laboratory of Prof Leo Spyracopoulos at the University of Alberta, where he studied the molecular mechanisms of polyubiquitin chain synthesis and their recognition by partner proteins in the DNA damage response using a combination of protein NMR, biochemical assays, and mathematical modelling. He subsequently carried out postdoctoral research at Stanford University with Prof Dan Herschlag and Prof Polly Fordyce, where he developed HT-MEK. He started his independent research group in the Manchester Institute of Biotechnology at The University of Manchester in 2023. His group focuses on developing and deploying state-of-the-art technologies for high-throughput, quantitative biochemistry to map the relationships between sequence, structure, and function in proteins and enzymes at a scale previously inaccessible.