News and Publications

Synthetic Protein Chemistry

P.E. Dawson, G. Beligere, T. Doundoulakis, T. M. Hackeng

The focus of our laboratory is the development and use of methods to incorporate unnatural chemical groups into proteins. Most efforts to understand the molecular basis for protein function have used biological techniques to modify the sequence of amino acids that defines a protein's structure and function. These modifications are limited to the naturally occurring 20 amino acids. We have 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 of up to about 200 amino acids. 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. Our goal is to introduce noncoded amino acids and other amino acid--like chemical groups into proteins in order to better understand the molecular basis of protein function.

An example of our synthetic approach for studying proteins is our work on human secretory phospholipase A₂ (hsPLA₂). This protein is a 14-kD lipolytic enzyme (124 residues, 7 disulfide bonds) with unknown physiologic function that is present in the -granules of blood platelets. The enzyme catalyzes the stereospecific hydrolysis of the fatty acid side-chain ester bond at the 2 position of phospholipids in a calcium-dependent reaction. Recently, studies have indicated that independent of its lipolytic activity, hsPLA₂ can inhibit prothrombinase-mediated blood coagulation. To separate the lipolytic activity from any possible binding activities, we designed a chemical analog in which the catalytic histidine-47 was replaced with the isosteric, but hydrogen bonding--deficient, amino acid thienylalanine.

To synthesize hsPLA₂, we used a ligation chemistry that joins unprotected peptides at X-Cys sites in the polypeptide sequence. The presence of 14 cysteine residues in hsPLA₂ allowed us to assemble the full-length protein from 4 synthetic fragments. After assembly, the protein was folded to form 7 disulfide bonds and assayed for enzymatic activity. As expected, hsPLA₂ with the native histidine was fully active, whereas the thienylalanine analog had no measurable lipolytic activity. We are using this construct to analyze the role of hsPLA₂ in blood coagulation.

Another objective in the laboratory is to understand the role of the polypeptide backbone in protein stability and ligand-substrate binding. To probe the hydrogen-bonding characteristics of the polypeptide backbone, we are using a chemical analog of the peptide bond that removes specific hydrogen-bonding interactions while maintaining the steric properties of the natural amide bond. We are using these substitutions to investigate the role of hydrogen bonding on the formation of -helices and stability in a coiled coil and in a mixed ß-sheet---helix protein. In addition, we are using this substitution to analyze a series of putative hydrogen-bonding interactions involved in the binding of peptide substrates by serine proteases such as trypsin and t-plasminogen activator.

PUBLICATIONS

Dawson, P.E., Churchill, M.J., Ghadiri, M.R., Kent, S.B.H. Modulation of reactivity in native chemical ligation through the use of thiol additives. J. Am. Chem. Soc. 119:4325, 1997.

Dawson, P.E., Fitzgerald, M.C., Muir, T.W., Kent, S.B.H. Methods for the chemical synthesis and readout of self-encoded arrays of polypeptide analogues. J. Am. Chem. Soc. 119:7917, 1997.

Hackeng, T.M., Mounier, C.M., Bon, C., Dawson, P.E., Griffin, J.H., Kent, S.B.H. Total chemical synthesis of enzymatically active human type-II secretory phospholipase A₂. Proc. Natl. Acad. Sci. U.S.A. 94:7845, 1997.