Protein engineering to accelerate scientific discovery
Currently we are working to develop generalizable protein engineering-based methods to facilitate protein structure determination by X-ray crystallography. We are also working to develop methods to re-engineer radical SAM enzymes to catalyze arbitrarily-chosen radical-mediated chemical reactions.
Moody laboratory approach
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 (PFLAEH, a radical SAM enzyme), for facile crystallization.2, 3 We observed that while PFLAEH 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 to greatly accelerate scientific discovery and disease treatment.
PFLAEH in its crystal lattice
4 distinct PFLAEH lattices share a screw axis
Synthetic screw axes could pre-order proteins of interest
In addition to PFLAEH, we are also investigating the sterile alpha motif (SAM) domain of human translocation Ets leukemia protein (TEL). The SAM domain of TEL spontaneously forms a 6-fold helical polymer. In 2007, researchers re-engineered the polymer subunits to form 3-fold helical polymers and to only polymerize at low pH. They then fused proteins of unknown structure to these polymer subunits and were able to reproducibly obtain crystals at low pH. Unfortunately, most of the crystals were too disordered to give usable X-ray diffraction data.4 We aim to reengineer the TEL-SAM subunits so that polymerization occurs in a more controlled manner, allowing the formation of ordered crystals.
The TEL-SAM polymer (magenta) fused to lysozyme (cyan)
While many other types of enzymes have been designed or re-designed through computational or experimental enzyme engineering, radical SAM enzymes have yet to be explored as rationally-engineered synthetic catalysts. We are computationally engineering radical SAM enzymes to accept novel substrates. 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.5
Radical SAM activation mechanism
Proposed enzyme-catalyzed radical cyclization reaction mechanism
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! We welcome dedicated, hardworking 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.
- Dale GE, Oefner C, D'Arcy A. The protein as a variable in protein crystallization. J Struct Biol. 2003 Apr;142(1):88-97.
- Moody JD, Shisler K, Horitani M, Lawrence M, Galambas A, Whittenborn E, Hoffman B, Drennan C, Broderick JB. The structural basis of [4Fe4S] valence localization in pyruvate formate-lyase activating enzyme. Manuscript in Preparation.
- Moody JD, Lawrence M, Whittenborn E, Drennan C, Broderick JB. Cis-peptides are structurally conserved in a subset of radical SAM enzymes. Manuscript in Preparation.
- Nauli S, Farr S, Lee YJ, Kim HY, Faham S, Bowie JU. Polymer-driven crystallization. Protein Science. 2007 Nov;16(11):2542-51.
- Broderick JB, Duffus BR, Duschene KS, Shepard EM. Radical S-adenosylmethionine enzymes. Chem Rev. 2014 Apr 23;114(8):4229-317.