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The Skaggs Institute For Chemical Biology
Scientific Report 1999-2000


Bioorganic Chemistry of Proteins


P. Dawson, G. Beligere, J. Blankenship, A. Brik, U. Manjappara, J. Offer, L. Yan

Our laboratory focuses on the development and use of methods of incorporating unnatural chemical groups into proteins. We developed a chemical approach for the production of the large polypeptide chains that make up protein molecules, enabling us to change the structure of a protein in ways impossible by natural means. We use solid-phase peptide synthesis to generate peptides up to about 50 amino acids long and then use chemoselective reactions to assemble the peptides into proteins up to about 150 amino acids long.

This "chemical ligation" approach greatly facilitates the synthesis of proteins of moderate size and has opened the world of proteins to the synthetic tools of organic chemistry. Chemical ligation can be extended to biologically expressed proteins, enabling the production of semisynthetic proteins of unlimited size that contain fluorophores or cross-linking agents at defined positions. Our goal is to introduce noncoded amino acids and other chemical groups into proteins to better understand the molecular basis of protein function.

Synthesis of Proteins

Support from the Skaggs Institute has enabled us to improve the methods for synthesizing proteins. The assembly of large proteins requires repeated ligation, deprotection, and purification steps that reduce the yield of the synthesis. We developed an approach for assembling the peptides on a solid support that enables us to rapidly synthesize proteins from multiple peptides. In addition to improving yields, solid-phase ligation will enable us to assemble large numbers of protein analogs in parallel.

A second area of research is the development of new ligation chemistries. We developed a benzyl scaffold that can be attached to the N-terminus of a peptide that will promote efficient peptide ligation in aqueous solution (Fig. 1). Future efforts will focus on improving the rate of ligation and on removing the scaffold from the peptide after ligation.

Oxidoreductases

Oxidoreductases, a class of highly conserved enzymes that catalyze the reduction and oxidation of disulfide bonds in proteins, are coupled to the cellular protein-folding machinery. The oxidoreductases share a common a/ß fold and a common active site consisting of Cys-Xaa-Yaa-Cys, yet have widely different oxidation potentials. Thioredoxin (108 amino acids) and glutaredoxin-3 (83 amino acids) are excellent candidates for analysis via total chemical synthesis. Incorporation of unnatural amino acids into these proteins will enable us to probe the secondary determinants of oxidation potential and to gain insights into the mechanism of reduction of disulfide bonds.

Protein Scaffolds For Enzymatic Catalysis

Many enzymes have a common 3-dimensional fold despite having large differences in their substrate specificity and mechanism. We are using chemical synthesis to introduce unnatural structural elements and functional groups into the oxidoreductase fold in order to design new protein-based catalysts. In a similar study, done in collaboration with E. Keinan, the Skaggs Institute, and F. Grynszpan, The Scripps Research Institute, we are using a small hexameric enzyme as a scaffold to generate novel amine-catalyzed reactions.

Protein Topology

Proteins are composed of linear polypeptide chains that fold to a defined 3-dimensional structure. We are interested in altering this linear topology by using chemical ligation to cyclize the polypeptide chain. Cyclization of a peptide may alter the folding properties by causing changes in the amino acid connectivity. Recently, we synthesized an interlocked protein based on the tetramerization domain of p53. The folding and stability properties of this protein catenane are being analyzed. We hope to extend this work into the synthesis of interlocked chains of proteins that can assemble into defined planar or 3-dimensional topologically linked materials.

Publications

Beligere, G.S., Dawson, P.E. Synthesis of a three zinc finger protein, Zif268, by native chemical ligation. Biopolymers 51:363, 1999.

Brik, A., Keinan, E., Dawson P.E. Protein synthesis by solid-phase chemical ligation using a safety catch linker. J. Org. Chem. 65:3829, 2000.

Cho, S., Dawson, P.E., Dawson, G. In vitro depalmitoylation of neurospecific peptides: Implication for infantile neuronal ceroid lipofuscinosis. J. Neurosci. Res. 59:32, 2000.

Deniz, A.A., Laurence, T.A., Beligere, G.S., Dahan, M., Martin, A.B., Chemla, D.S., Dawson, P.E., Schultz, P.G., Weiss, S. Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Proc. Natl. Acad. Sci. U. S. A. 97:5179, 2000.

King, C.C., Zenke, F.T., Dawson, P.E., Dutil, E.M., Newton, A.C., Hemmings, B.A., Bokoch, G.M. Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1. J. Biol. Chem. 275:10108, 2000.

Marx, P.F., Hackeng, T.M., Dawson, P.E., Griffin, J.H., Meijers, J.C., Bouma, B.N. Inactivation of active thrombin-activatable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage. J. Biol. Chem. 275:12410, 2000.

Offer, J., Dawson, P.E. Nα-2-Mercaptobenzylamine-assisted chemical ligation. Org. Lett. 1:23, 2000.

 

 







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