Stephen Kent invents synthetic chemistries and uses them to probe the molecular basis of protein function.
Research Interests:
My research is focused on understanding the molecular basis of protein function, particularly the physical organic chemistry of enzyme catalysis. To that end, we develop novel methods for the total synthesis of proteins that enable us to apply advanced physical methods in unprecedented ways to understand the chemical origins of protein structure and function. We then demonstrate that knowledge by the design and construction of protein molecules with novel properties.
Chemical Protein Synthesis
The Kent research group pioneered the total chemical synthesis of protein molecules based on modern chemical ligation methods. The long polypeptide chains of protein molecules are prepared by convergent synthesis using chemical ligation – chemoselective condensation of unprotected peptide segments that contain unique, mutually reactive functional groups. We developed the most powerful ligation chemistry, native chemical ligation – thioester-mediated, amide-forming regioselective ligation at Xaa-Cys sites. The resulting polypeptide chains are folded with great efficiency to give high purity synthetic proteins. The covalent structure of synthetic proteins is confirmed by mass spectrometry, and three-dimensional folded structures are determined at atomic resolution by X-ray crystallography. Synthetic proteins have full biochemical and biological activities. Chemical protein synthesis is generally applicable to the efficient preparation of protein molecules > 30 kDa, with polypeptide chains containing 300 or more amino acids.
Total synthesis enables the versatile modification and labeling of protein molecules without restriction as to the sites, numbers, or kinds of chemical moieties being introduced.
Racemic Protein Crystallography
Total chemical synthesis enables us to make mirror image D-protein molecules not found in nature. We have pioneered the use of racemic & quasi-racemic protein crystallography to determine the X-ray structures of proteins that otherwise will not crystallize, and to obtain electron density maps of unprecedented quality.
Mirror Image Drug Discovery
In collaboration with Sachdev Sidhu (Toronto) we pioneered the use of mirror image protein targets prepared by total chemical synthesis to generate affinity-matured binders from phage-displayed protein libraries. Chemical synthesis of the identified binders as D-protein molecules of the same amino acid sequence gives synthetic D-protein binders that have the same high affinity for the natural target protein. These D-proteins have the specificity and potency of antibodies, are non-immunogenic, non-toxic, resistant to natural proteases, long-lived in vivo, and show great promise as candidate human therapeutics.
Selected Publications
Characterization of protein molecules prepared by total chemical synthesis. Stephen B.H. Kent, in: Ashfraf Brik, Philip E. Dawson, Lei Liu (Eds.), Total Chemical Synthesis of Proteins. ISBN: 978-3-527-34660-8, Wiley-VCH; 1st edition (March 22, 2021).
Chemical synthesis of an enzyme containing an artificial catalytic apparatus. Vladimir Torbeev, Stephen B.H. Kent. Aust. J. Chem. 2020; 73:321–326.
Novel protein science enabled by total chemical synthesis. Kent SBH, Protein Science 2019; 28:313–328.
Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis. Kent SBH, Current Opinion in Chemical Biology 2018; 46:1–9.
Inversion of Thr and Ile side chain stereochemistry in a protein molecule: impact on the folding, stability, and structure of the ShK toxin protein molecule,. Bobo Dang, Tomoya Kubota, Rong Shen, Kalyaneswar Mandal, Francisco Bezanilla, Benoit Roux, Stephen B. H. Kent, Angewandte Chemie Int. Ed. 2017; 56:3324-3328.
Chemical protein synthesis: inventing synthetic methods to decipher how proteins work. Kent SBH. Bio Org Med Chem. 2017; 25:4926-4937.
Perplexing cooperative folding and stability of a low sequence complexity, poly-proline 2 protein lacking a hydrophobic core. Zachary Gates, Michael C Baxa, Wookyung Yu, Joshua A Riback, Hui Li, Benoit Roux, Stephen Kent, Tobin R Sosnick, Proc.Nat.Acad.Sci. 2017; 114:2241-2246.
A potent D-protein antagonist of VEGF-A is non-immunogenic, metabolically stable and longer-circulating in vivo. Maruti Uppalapati, Dong Jun Lee, Kalyaneswar Mandal, Hongyan Li, Les P. Miranda, Joshua Lowitz, John Kenney, Jarrett J. Adams, Dana Ault-Riché, Stephen B. H. Kent, Sachdev S. Sidhu, ACS Chemical Biology 2016; 11:1058-1065.