The Skaggs Institute
for Chemical Biology

Scientific Report 2005

Synthetic Protein Chemistry

P.E. Dawson, F. Brunel, M. Churchill, N. Metanis, T. Tiefenbrunn

We focus on using synthetic chemistry to understand the molecular basis of protein structure and function. Total chemical synthesis facilitates the fine-tuning of amino acid side chains to achieve, for example, greater conformational stability or to alter the binding specificity of proteins. Currently, we are developing therapies and potential vaccine candidates for HIV disease and are doing research to understand the molecular basis of enzymatic catalysis.

Selenoglutaredoxin

Glutaredoxin is an oxidoreductase with homology to the thioredoxins and protein disulfide isomerases. These oxidoreductases are critical regulators of redox potential and the formation of disulfide bonds in cells. Oxidoreductases use a conserved CXXC motif to tune the redox potential of the active site. Glutaredoxin functions as a hydride donor for ribonuclotide reductase. We are using chemical synthesis to introduce the amino acid selenocysteine into the active site of glutaredoxin to investigate the effects of forming sulfur-selenium or selenium-selenium bonds on the redox potential of this protein. In addition, we anticipate that the mixed selenium-sulfur enzymes will gain significant peroxidase activities. Using chemical ligation strategies, we have now synthesized a series of glutaredoxin analogs, and we are characterizing their structures and functions.

HIV Vaccine Design

Eliciting broadly neutralizing antibodies is a major goal in HIV vaccine design. This effort is complicated by the poor accessibility of conserved regions of HIV envelope proteins to antibodies. The membrane proximal region of the HIV envelope protein gp41 has generated marked research because of the discovery of 2 neutralizing antibodies that bind this sequence. We are collaborating with D.R. Burton, Scripps Research, and I.A. Wilson, the Skaggs Institute, to design peptides that mimic the helical conformation of the peptide that binds to the neutralizing antibody 4E10.

Immunization with simple peptides obtained from gp41 elicit a nonneutralizing response, presumably because of differences between the neutralizing and the most immunogenic conformations of the peptide. We fully characterized the 4E10 epitope and constrained the peptide to adopt the primarily helical conformation recognized by the 4E10 antibody. These studies have yielded a series of peptidomimetics that bind as tightly to the 4E10 antibody as the full-length gp41 glycoprotein does. Our goal is to eliminate all surfaces of the peptide that are not required for 4E10 binding or to mask them with soluble polymers and carbohydrates. We plan to use this negative design approach in combination with binding studies, immunization, and structural analysis by crystallography.

Protein-Carbohydrate Interactions

Glycosylation of the HIV coat proteins is critical for viral stability and protection of the virus from the immune system. Recent research indicated that rare oligosaccharides rich in mannose on this virus are involved in molecular interactions with human proteins such as DC-SIGN that are critical for HIV infection. These oligosaccharides are recognized by HIV-neutralizing proteins from cyanobacteria; one of these proteins, cyanovirin, is in clinical trials as an HIV microbicide. We are developing a total synthesis of the gp120-binding proteins DC-SIGN and cyanovirin to enable detailed structure-function studies on their carbohydrate-binding properties and their potential as HIV-inhibiting pharmaceuticals. In particular, efforts will be directed toward improving affinity and stability. Finally, we are interested in developing polyvalent dendrimers of these domains to increase multivalent interactions with the HIV coat glycoproteins.

Publications

Beltran, A.C., Dawson, P.E., Gottesfeld, J.M. Role of DNA sequence in the binding specificity of synthetic basic-helix-loop-helix domains. Chembiochem 6:104, 2004.

Brunel, F.M., Dawson, P.E. Synthesis of constrained helical peptides by thioether ligation: application to analogs of gp41. Chem. Commun. (Camb.) 2552, 2004, Issue 20.

Deechongkit, S., Dawson, P.E., Kelly, J.W . Toward assessing the position-dependent contributions of backbone hydrogen bonding to β-sheet folding thermodynamics employing amide-to-ester perturbations. J. Am. Chem. Soc. 126:16762, 2004.

Kamikubo, Y., De Guzman, R., Kroon, G., Curriden, S., Neels, J.G., Churchill, M.J., Dawson, P., Oldziej, S., Jagielska, A., Scheraga, H.A., Loskutoff, D.J., Dyson, H.J. Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin. Biochemistry 43:6519, 2004.

McNulty, J.C., Jackson, P.J., Thompson, D.A., Chai, B., Gantz, I., Barsh, G.S., Dawson, P.E., Millhauser, G.L. Structures of the agouti signaling protein. J. Mol. Biol. 346:1059, 2005.

Metanis, N., Keinan, E., Dawson P.E. A designed synthetic analog of 4-OT is specific for a non-natural substrate. J. Am. Chem. Soc.127:5862, 2005.