News and Publications

Bioorganic Chemistry of Proteins

Synthetic Protein Chemistry

Our laboratory focuses on the bioorganic chemistry of proteins. Proteins are large organic molecules of amazing compositional and conformational diversity. Through proteins the information stored in the DNA sequence of the genome is given functional form; consequently, all cellular processes are mediated through the actions of proteins. Efforts to understand the molecular basis for protein function have largely used biological techniques to modify the sequence of amino acids that defines a protein's structure and function. Unfortunately, these modifications are limited to the naturally occurring 20 amino acids.

We have developed a chemical approach for producing the large polypeptide chains that make up protein molecules. Synthetic access to these polypeptides enables us to change the structure of a protein in ways impossible in Nature. We use solid-phase peptide synthesis to generate peptides up to approximately 50 amino acids long and then use chemoselective reactions to assemble the peptides into proteins up to about 200 amino acids long. This chemical ligation approach greatly facilitates the synthesis of proteins of moderate size and has opened up the world of proteins to the synthetic tools of organic chemistry (Fig. 1A).

One of our goals is to develop more efficient methods for protein synthesis. Recently, we extended the native chemical ligation technique by increasing the number of ligation sites that can be used and by simplifying the synthesis of Cα-thioester peptides used in the ligation reaction. Other studies focus on using the structural information stored in the amino acid sequence of peptide fragments. Many proteins can adopt a structure similar to the native protein even when their polypeptide chains are broken into 2 or more fragments. This peptide self-assembly has enabled us to use regioselective chemical methods to synthesize proteins. Conformationally assisted ligation will enable us to synthesize a wider range of proteins with few limitations on the peptide sequence (Fig. 1B).

Backbone Interactions In Protein Stability and Folding

We are using our ability to chemically manipulate proteins to probe some fundamental issues in protein science. One of the greatest challenges in the biochemical sciences is to understand the forces and mechanisms that guide a linear polypeptide into its functional folded form. We are investigating the role of the polypeptide backbone in these processes. Although it accounts for about 50% of the mass of a protein, the backbone cannot be modified by standard biological methods. In our studies, we are replacing the natural amide bond in the polypeptide backbone with an ester bond. This replacement deletes 2 hydrogen bonds while keeping the conformational properties of the natural amide backbone. We selected 2 protein domains: the coiled coil from the transcription factor GCN4 and a protein-based serine protease inhibitor, CI2. Both of these proteins have a prominent α-helix that is an essential component of their 3-dimensional structure. Systematic incorporation of ester bonds into these well-studied proteins will give new insight into the role of backbone hydrogen-bonding interactions in the formation and stability of α-helical structures in proteins.

Publications

Beligere, G.S., Dawson, P.E. Conformationally assisted protein ligation using C-terminal thioester peptides. J. Am. Chem. Soc. 121:6332, 1999.

Coombs, G.S., Rao, M.S., Olson, A.J., Dawson, P.E. Revisiting catalysis by chymotrypsin family serine proteases using peptide substrates and inhibitors with unnatural mainchains. J. Biol. Chem. 274:24074, 1999.

Hackeng, T.M., Griffin, J.H., Dawson, P.E. Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology. Proc. Natl. Acad. Sci. U. S. A. 96:10068, 1999.