The Faculty

Focus

Chemical protein synthesis plus advanced physical methods to elucidate the molecular basis of protein biological function

Biography

Born Wellington, New Zealand 1945.
Victoria University, B.Sc., 1968.
Massey University, M.Sc., 1970.
University of California, Berkeley, Ph.D., 1975.
The Rockefeller University, Research Associate 1974-1977, Assistant Professor 1977-1981.
The California Institute of Technology, Senior Research Associate 1983-1989.
Bond University, Professor 1989-1990.
The Scripps Research Institute, Member & Professor, 1991-1996.
Gryphon Sciences, Chief Scientist, 1997-2000.
The University of Chicago, Professor, 2001-.
Director, Institute for Biophysical Dynamics, 2003-2009.
Joint appointment with the Department of Biochemistry & Molecular Biology.

Accolades

2011 Bader Award in Bioorganic Chemistry, American Chemical Society.
2010 Akabori Medal, Japanese Peptide Society.
2010 Rudinger Award, European Peptide Society.
2009 R. Bruce Merrifield Award, American Peptide Society.
2008 Fellow, Royal Society of Chemistry.
2006 Honorary Fellow, Royal Society of New Zealand.
2004 Vincent duVigneaud Award, American Peptide Society.
2002 E.T. Kaiser Jr. Award for Innovation in Protein Science, The Protein Society.
2000 Fellow, American Association for the Advancement of Science.
1994 Hirschmann Award in Peptide Chemistry, American Chemical Society.
1968 Senior Scholar, Victoria University.

Research Interests

The Kent research group is devoted to inventing and using new chemistries to reveal how proteins work in nature. 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 basis of protein function. Our goal is to then demonstrate that knowledge by the design and construction of protein molecules with novel properties.

Chemical Protein Synthesis
The total synthesis of natural products is arguably the most important intellectual endeavor in the area of synthetic organic chemistry - it drives methodology forward and in the process generates new molecular entities. Proteins are the most abundant, exciting, and challenging class of natural products. Proteins are the molecular machines of the living world, performing nearly all of the functions in the cell and playing vital roles in human biology and medicine. With the success of the genome sequencing projects, proteins are being discovered at an unprecedented rate – a single recent publication described several million novel protein molecules, known only as open reading frame data encoded in the genomic DNA. In addition, it has been estimated that there are more than one million venom-derived proteins, each of which has potent and specific biological activities.

The robust synthesis of protein molecules was one of the ‘grand challenges’ of organic chemistry in the twentieth century. Our laboratory invented the chemical ligation methods that met this challenge, and that have enabled the practical total synthesis of proteins. Chemical protein synthesis is generally applicable to the efficient preparation of polypeptides containing 300 or more amino acids. Total synthesis enables the versatile incorporation of non-coded amino acids into proteins, and the modification and labeling of protein molecules, without restriction as to the sites, numbers, and kinds of chemical moieties being introduced.

We prepare the long polypeptide chains of protein molecules by the chemoselective reaction (‘chemical ligation’) of unprotected peptide segments containing unique, mutually reactive functional groups. The most powerful of these ligation chemistries is thioester-mediated, amide-forming ligation, termed ‘native chemical ligation’. The resulting polypeptide chains are then folded with great efficiency to give high purity synthetic proteins. The covalent structure of the molecule is confirmed by mass spectrometry and the three dimensional folded structure of the synthetic protein is determined by Xray crystallography.

Chemical Protein Synthesis

Recently, we introduced ‘kinetically controlled ligation’, a novel and highly sophisticated chemistry for the fully convergent synthesis of large protein molecules. Currently we are exploring novel insertion reactions for the creation of molecular diversity in preformed molecular scaffolds, and the use of polymer-supported ligation chemistries for the synthesis of proteins.

Chemistry of Enzyme Catalysis
We take full advantage of the flexibility provided by total protein synthesis to study the physical organic chemistry of how enzyme molecules work. Backbone engineering of the amide bonds in the polypeptide chain has been used to delete critical H-bonding interactions and to evaluate the effects on enzyme function. 13C & 15N NMR probe nuclei have been introduced at unique single atom sites to elucidate critical aspects of the chemical basis of enzyme catalysis. Single molecule fluorescence spectroscopy is being used to probe the functional properties of uniquely chemical analogues of the enzyme molecule.

Mirror Image Protein Molecules
The chemical synthesis approach enables us to make enantiomeric ‘D-protein’ molecules not found in nature. We have pioneered the use of racemic protein crystallography to determine the Xray structures of proteins that will otherwise not crystallize, and to obtain protein electron density maps of unprecedented accuracy.

Mirror image enzyme HIV-1 protease

The mirror image enzyme HIV-1 protease prepared by total chemical synthesis
{Image credit: Art Olson, TSRI}

We are also prototyping ‘mirror image drug discovery’, the use of mirror image protein targets to identify novel lead compounds from protein libraries. Synthesis of the enantiomer of the identified protein binders gives unique molecules, that could not have been discovered using the natural target, with the specificity and potency of antibodies and effective against the protein found in nature. The resulting D-protein therapeutic candidates are small enough to be made by existing chemical methods of production.

Selected References

  1. Design, total chemical synthesis, and X-ray structure of a protein having a novel polypeptide chain topology. Kalyaneswar Mandal, Brad L. Pentelute, Duhee Bang, Zachary P. Gates, Vladimir Yu. Torbeev, Stephen B. H. Kent, Angewandte Chem Int Ed, 51, 1481-1486 (2012).
  2. Single-Molecule Studies of HIV-1 Protease Catalysis Enabled by Chemical Protein Synthesis. Vladimir Yu. Torbeev, Sua Myong, Taekjip Ha, Stephen B.H. Kent, Israel J. Chem., 51, 960-967 (2011).
  3. Protein conformational dynamics in the chemical mechanism of HIV-1 protease catalysis. Vladimir Yu. Torbeev, H. Raghuraman, Donald Hamelberg, Marco Tonelli, William M. Westler, Eduardo Perozo & Stephen B. H. Kent, Proc Natl Acad Sci USA, 108, 20982-20987 (2011). PMC3248522
  4. Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography. Brad L. Pentelute, Kalyaneswar Mandal, Zachary P. Gates, Michael R. Saway, Todd O. Yeates, Stephen B. H. Kent, Chem. Commun., 46, 8174 - 8176 (2010).
  5. Determination of the X-ray structure of the snake venom protein Omwaprin by total chemical synthesis and racemic protein crystallography. James R. Banigan, Kalyaneswar Mandal, Michael R. Sawaya, Vilasak Thammavongsa, Antoni Hendrickx Olaf Schneewind, Todd Yeates, Stephen Kent, Protein Science, 9, 1840-1849 (2010).  PMC2998720
  6. A semisynthesis platform for investigating structure-function relationships in the N-terminal domain of the anthrax lethal factor. Brad L. Pentelute, Adam P. Barker, Blythe E. Janowiak, Stephen B. H. Kent, R. John Collier, ACS Chemical Biology, 5, 359-64 (2010).  PMC2855745
  7. A one-pot approach to neoglycopeptides using orthogonal native chemical ligation and click chemistry. Dong Jun Lee, Kalyaneswar Mandal, Paul W. R. Harris, Margaret A. Brimble, and  Stephen B. H. Kent, Organic Letters, 11, 5270-3 (2009).
  8. Biomimetic synthesis of lispro insulin via a chemically synthesized ‘mini-proinsulin’ prepared by oxime-forming ligation. Youhei Sohma, Stephen B. H. Kent, J Am Chem Soc, 131, 16313-8 (2009).
  9. Kochendoerfer GG, {25 co-authors} Kent SB, Bradburne JA.  Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein. Science 2003; 299:884-7.
  10. Torbeev VY, Kent SBH. Convergent chemical synthesis and crystal structure of a 203 amino acid ‘covalent dimer’ HIV-1 protease enzyme molecule.  Angew Chem Int Ed 2007; 46:1667-70.
  11. Durek T, Torbeev VY, Kent SBH. Convergent chemical synthesis and high resolution X-ray structure of human lysozyme. PNAS USA 2007; 104:4846-51. PMC1829227
  12. Kent SBH. Total chemical synthesis of proteins. Chemical Society Reviews 2009; 38:338-51.
  13. Ionization state of the catalytic dyad Asp25/25’ in the HIV-1 protease: NMR studies of site-specifically 13C labeled HIV-1 protease prepared by total chemical synthesis. Vladimir Yu. Torbeev, Stephen B. H. Kent, Organic & Biomolecular Chemistry, 10, 5887–5891 (2012).PMCID 3437676
  14. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus VEGF-A} protein complex by racemic crystallography.  Kalyaneswar Mandal, Maruti Uppalapati, Dana Ault-Riché, John Kenney, Joshua Lowitz, Sachdev Sidhu*, Stephen B.H. Kent*, Proc Natl Acad Sci USA, 109, 14779-14784 (2012).PMCID 3443191
  15. Fully convergent chemical synthesis of ester insulin: determination of the high resolution X-ray structure by racemic protein crystallography. Michal Avital-Shmilovici, Kalyaneswar Mandal, Zachary P. Gates, Nelson Phillips, Michael A. Weiss, Stephen B.H. Kent, J. Am. Chem. Soc., 135, 3173–3185 (2013). PMCID3625376