James Moody

Research: Protein engineering to accelerate scientific discovery.

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Currently we are working to develop generalizable protein engineering-based methods to facilitate protein structure determination by X-ray crystallography.

 X-ray crystallography allows us to determine the structure of proteins at the atomic level, helping us to understand how protein dysfunction causes disease, develop new treatments, and engineer new protein-based tools. Unfortunately, X-ray crystallography is only useful for those proteins that can be induced to form ordered crystals; about 20-30% of all known proteins.1 Recently we engineered a variant of pyruvate formate-lyase activating enzyme (PFL-AE-H), a radical SAM enzyme, for facile crystallization. We observed that while the engineered PFL-AE variant formed at least 4 different crystal packing arrangements (lattices), all of these shared a conserved screw axis. Screw axes can be thought of as ordered fibers composed of stacked copies of the protein. Since this screw axis is common among 4 different crystal lattices, we propose that it forms first during crystal nucleation and serves to dictate the packing arrangement of the rest of the crystal. Currently we are investigating fusing proteins of interest to engineered screw axis fibers to pre-order them and nucleate crystal formation. It is our hope that new protein crystallization methods like this one will enable structure determination of a much greater percentage of known proteins and greatly accelerate scientific discovery and disease treatment.    

In the Moody lab you will learn computational protein modeling and design, molecular biology techniques, protein biochemistry, and macromolecular X-ray crystallography. If you’re interested, I’d love to talk with you! I welcome dedicated, hard working students with all levels of experience, including beginning students. Be prepared to dedicate at least 10 hours per week to the research to make meaningful progress.

To learn more about radical SAM enzymes and some of the first proteins we aim to crystallize, keep reading!

Radical SAM enzymes create highly reactive organic radicals and use them to accomplish a huge variety of high-energy chemical transformations in substrate molecules, nucleic acids, and other proteins.2

 Right now we’re studying the interactions between a radical SAM enzyme, glycerol dehydratase activating enzyme (GD-AE), and its substrate, B12-independent glycerol dehydratase (GD).2 We’ve modeled the complex and are working to determine its atomic structure using chemical crosslinking, mass spectrometry, protein engineering, and crystallography. This will help us understand the role and mechanism of the unusual ferredoxin domain of GD-AE and the mechanism by which the GD substrate peptide receives the radical modification and is re-inserted into the GD active site.

 Using the same approaches we’re also studying the interactions between the FeFe-hydrogenase maturases HydE, HydF, and HydG. Structural characterization of complexes of these enzymes will provide clues to the identity of the elusive HydE substrate and the mechanism by which these maturases assemble the complex 2-iron subcluster of FeFe-hydrogenase.2