News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
Bioorganic Chemistry of Proteins
P.E. Dawson, G. Beligere, J. Blankenship, A. Brik, J. Offer
Synthetic Protein Chemistry
Our laboratory focuses on the bioorganic chemistry of proteins. Proteins are
large organic molecules of amazing compositional and conformational diversity.
Through proteins the information stored in the DNA sequence of the genome is
given functional form; consequently, all cellular processes are mediated through
the actions of proteins. Efforts to understand the molecular basis for protein
function have largely used biological techniques to modify the sequence of amino
acids that defines a protein's structure and function. Unfortunately, these modifications
are limited to the naturally occurring 20 amino acids.
We have developed a chemical approach for producing the large polypeptide
chains that make up protein molecules. Synthetic access to these polypeptides
enables us to change the structure of a protein in ways impossible in Nature.
We use solid-phase peptide synthesis to generate peptides up to approximately
50 amino acids long and then use chemoselective reactions to assemble the peptides
into proteins up to about 200 amino acids long. 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 (Fig. 1A).
One of our goals is to develop more efficient methods for protein synthesis.
Recently, we extended the native chemical ligation technique by increasing the
number of ligation sites that can be used and by simplifying the synthesis of
Cα-thioester peptides used in the ligation reaction. Other studies focus
on using the structural information stored in the amino acid sequence of peptide
fragments. Many proteins can adopt a structure similar to the native protein
even when their polypeptide chains are broken into 2 or more fragments. This
peptide self-assembly has enabled us to use regioselective chemical methods to
synthesize proteins. Conformationally assisted ligation will enable us to synthesize
a wider range of proteins with few limitations on the peptide sequence (Fig.
Backbone Interactions In Protein Stability and Folding
We are using our ability to chemically manipulate proteins to probe some fundamental
issues in protein science. 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 accounts for about 50% of the mass of
a protein, the backbone cannot be modified by standard biological methods. In
our studies, we are replacing the natural amide bond in the polypeptide backbone
with an ester bond. This replacement deletes 2 hydrogen bonds while keeping the
conformational properties of the natural amide backbone. We selected 2 protein
domains: the coiled coil from the transcription factor GCN4 and a protein-based
serine protease inhibitor, CI2. Both of these proteins have a prominent α-helix
that is an essential component of their 3-dimensional structure. Systematic incorporation
of ester bonds into these well-studied proteins will give new insight into the
role of backbone hydrogen-bonding interactions in the formation and stability
of α-helical structures in proteins.
Beligere, G.S., Dawson, P.E. Conformationally assisted protein ligation
using C-terminal thioester peptides. J. Am. Chem. Soc. 121:6332, 1999.
Coombs, G.S., Rao, M.S., Olson, A.J., Dawson, P.E. Revisiting catalysis
by chymotrypsin family serine proteases using peptide substrates and inhibitors
with unnatural mainchains. J. Biol. Chem. 274:24074, 1999.
Hackeng, T.M., Griffin, J.H., Dawson, P.E. Protein synthesis by native
chemical ligation: Expanded scope by using straightforward methodology. Proc.
Natl. Acad. Sci. U. S. A. 96:10068, 1999.