News and Publications

Bioorganic Chemistry of Proteins

P.E. Dawson, J. Blankenship, C. Boddy, J.L. Offer, C. Neidre

Our focus is synthetic protein chemistry. We developed a set of highly selective chemical reactions that allow the short peptides available from solid-phase peptide synthesis to be assembled into single-domain and multidomain proteins. With these methods, we can incorporate unnatural amino acids to probe fundamental questions about protein folding, stability, and enzymatic catalysis. In addition, we can generate proteins with desired affinity and fluorescent labels or with photo cross-linking agents. We used these tools recently to study the prothrombinase complex and protein-protein interactions involving chemokines.

SYNTHESIS OF PROTEINS

We developed a simple chemical auxiliary that can be attached directly to the N terminus of a peptide. This auxiliary mimics the chemical properties of an N-terminal cysteine and facilitates a sulfur to nitrogen acyl transfer, forming a peptide bond at the ligation site. After acyl transfer, the auxiliary can be removed with acid, leaving a native peptide as the final product. This method promises to eliminate the need for cysteine residues and opens up nearly all proteins of moderate size (~150 amino acids) to total chemical synthesis. (Much larger proteins can be accessed through biological expression combined with chemical synthesis.) In addition, several posttranslational modifications such as ubiquitination may be accessible via these methods.

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 a combination of folding and chemical ligation to make structures called catenanes, which consist of 2 interlocked cyclic peptides. Such circularization and topological linking of a peptide is expected to alter the stability and folding properties of a protein.

To test these concepts, we synthesized an interlocked protein based on the tetramerization domain of p53 that is extremely stable to thermal denaturation (Fig. 1). This catenane is a good model for studies of bimolecular protein folding and assembly, and because the parent p53 protein is involved in tumor suppression, topological linking may be a new mechanism for controlling cell survival by stabilizing or destabilizing the natural protein. A future direction of this work is the synthesis of interlocked chains of proteins that can assemble into defined topologically linked materials.

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 designed inhibitors of this protein to facilitate the development of a model for infantile neuronal ceroid lipofuscinosis and explain the neuronal death that occurs in this disease. Recently, we found that these inhibitors increase the susceptibility of neuroblastoma-derived cells to apoptosis induced by chemotherapeutic agents such as etoposide.

PUBLICATIONS

Marinzi, C., Bark, S.J., Offer, J., Dawson, P.E. A new scaffold for amide ligation. Bioorg. Med. Chem. 9:2323, 2001.

Offer, J., Boddy, C.N., Dawson, P.E. Extending synthetic access to proteins with a removable acyl-transfer auxiliary. J. Am. Chem. Soc. 124:4642, 2002.

Yan, L.Z., Dawson, P.E. Design and synthesis of a protein catenane. Angew. Chem. Int. Ed. 40:3625, 2001.

Yegnéswaran, S., Fernández, J.A., Griffin, J.H., Dawson, P.E. Factor Va increases the affinity of factor Xa for prothrombin: a binding study using a novel photoactivable thiol-specific fluorescent probe. Chem. Biol. 9:485, 2002.