Chemical Ligation / Acyl Transfer

One goal of our laboratory is to develop methods to incorporate unnatural chemical groups into proteins. The chemical ligation approach utilizes solid phase peptide synthesis to generate several ~40 amino acid peptides which are then assembled using chemoselective reactions to make proteins up to 150 amino acids in length. Chemical ligation 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 semisynthesis of proteins of unlimited size that contain fluorophores or crosslinking agents at defined positions. Our general goal is to introduce non-coded amino acids and other chemical groups into proteins to better-understand the molecular basis of protein function.

Folding and Stability

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 comprises ~50% of the mass of a protein, the backbone of proteins cannot be modified by standard biological methods. As a result, most experimental work on protein folding has studied amino acid side chains by substituting alanine or glycine residues into the polypeptide sequence. In contrast, chemical synthesis allows the polypeptide backbone to be modified. For example, substitution of the backbone amide bond with an ester bond deletes two 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 small proteins such as chymotrypsin inhibitor 2.

Backbone Topology

Proteins are composed of linear polypeptide chains that fold to a defined three 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 due changes in the amino acid connectivity. We have 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 three dimensional toplogically linked materials.

Protein:Protein Interactions

We are interested in how proteins interact with other molecules in complex systems that are difficult to analyze at high resolution. Our approach is to make individual proteins by synthetic and semisynthetic methods that incorporate site-specific photo crosslinking agents flourophores and affinity tags. Following crosslinking, these proteins can be isolated along with any molecule that was in the proximity the crosslinking agent. These molecules can then be identified and mapped by mass spectrometry (MALDI and ESI). We have designed a multifunctional reagent that contains both a fluorescent group and a crosslinking agent. In collaboration with Dr. John Griffin we are using this reagent to study the prothrombinase complex. We are also using this approach in a collaboration with Dr. Dan Salomon to understand matrix:chemokine:chemokine receptor interactions and to develop a synthetic matrix for tissue engineering.

Protein Palmitoylation

The dynamic palmitoylation of proteins through thioester linkages on cysteine residues has been shown to be required for the regulation of many membrane associated proteins. Infantile neuronal ceroid lipofuscinosis (INCL) is believed to be a lysosomal storage disease and caused by mutations in the gene for palmitoyl protein thioesterase 1 (PPT1). In collaboration with Dr. Glyn Dawson, University of Chicago, we have designed inhibitors of this protein to facilitate the development of a model for INCL and explain the neuronal death in this disease. We have recently found that these inhibitors increase the susceptibil ity of neuroblastoma derived cells to apoptosis induced by chemotherapeutic agents such as etoposide. Future directions will include efforts to characterize the identity and mechanism of the lysosomal storage material and in the development of small molecule catalysts of palmitoyl thioester hydrolysis.