News and Publications

Bioorganic Chemistry of Proteins

P.E. Dawson, G. Beligere, J. Blankenship, A. Brik, U. Manjappara, C. Marinzi, J. Offer, L. Yan, N. Metanis

SYNTHETIC PROTEIN CHEMISTRY

Our laboratory focuses on the bioorganic chemistry of proteins. Proteins are large organic molecules of amazing compositional and conformational diversity. We developed a chemical approach for the production of the large polypeptide chains that make up protein molecules. Being able to synthesize these polypeptides allows us to change the structure of a protein in ways impossible by biological methods. 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 150 amino acids. 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.

BACKBONE INTERACTIONS IN PROTEIN STABILITY AND FOLDING

We are interested in 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. Consequently, most experimental work on protein folding has involved substituting alanine or glycine residues for other residues in amino acid side chains in the polypeptide sequence. In contrast, the chemical methods we developed allow modification of the polypeptide backbone. For example, replacement of the backbone amide bond with an ester bond deletes 2 hydrogen bonds while keeping intact the conformational properties of the natural amide backbone. We are using this approach to study the role of the backbone in the folding pathway of chymotrypsin inhibitor 2.

CHEMICAL CROSS-LINKING

We are also interested in how proteins interact with other molecules in complex systems that are difficult to analyze at high resolution. Our approach is to use synthetic and semisynthetic methods to incorporate light-triggered cross-linking fluorophores and affinity tags at specific sites in individual proteins. After cross-linking, these proteins can be isolated along with any biomolecule that was near the cross-linking agent. These biomolecules can then be identified and mapped by using mass spectrometry.

We designed a multifunctional reagent that contains both a fluorescent group and a cross-linking agent. In collaboration with J. Griffin, Department of Molecular and Experimental Medicine, we used this reagent to study the prothrombinase complex. These studies suggest that one mechanism by which coagulation factor Va enhances the activity of factor Xa toward prothrombin is by markedly increasing the affinity of factor Xa for prothrombin.

PALMITOYL PROTEIN THIOESTERASE

Infantile neuronal ceroid lipofuscinosis is caused by a deficiency in palmitoyl protein thioesterase. This enzyme removes palmitate from specific cysteine residues in proteins. In collaboration with G. Dawson, University of Chicago, we are designing inhibitors of this enzyme to facilitate the development of a model for infantile neuronal ceroid lipofuscinosis and explain the neuronal death that occurs in this disease.

PUBLICATIONS
Beligere, G.S., Dawson, P.E. Design, synthesis, and characterization of 4-ester C12, a model for backbone hydrogen bonding in protein a-helices. J. Am. Chem. Soc. 122:12079, 2000.

Cho, S., Dawson, P.E., Dawson, G. Antisense palmitoyl protein thioesterase 1 (PPT1) treatment inhibits PPT1 activity and increases cell death in LA-N-5 neuroblastoma cells. J. Neurosci Res. 62:234, 2000.

Dawson, P.E., Kent, S.B.H. Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 69:923, 2000.

Hackeng, T.M., Fernandez, J.A., Dawson, P.E., Kent, S.B., Griffin, J.H. Chemical synthesis and spontaneous folding of a multidomain protein: Anticoagulant microprotein S. Proc. Natl. Acad. Sci. U. S. A. 97:14074, 2000.

Pelletier, A.J., van der Laan, L.J.W., Hildbrand, P., Siani, M.A., Thompson, D.A., Dawson, P.E., Torbett, B.E., Salomon, D.R. Presentation of chemokine SDF-1 a by fibronectin mediates directed migration of T cells. Blood 96:2682, 2000.

Yan, L., Dawson, P.E. Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J. Am. Chem. Soc. 123:526, 2001.

Dawson Website