News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1999-2000
Bioorganic Chemistry of Proteins
P. Dawson, G. Beligere, J. Blankenship, A. Brik, U. Manjappara, J. Offer,
Our laboratory focuses on the development and use of methods of incorporating
unnatural chemical groups into proteins. We developed a chemical approach for
the production of the large polypeptide chains that make up protein molecules,
enabling us to change the structure of a protein in ways impossible by natural
means. 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 up to about 150 amino acids long.
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. Chemical ligation can be extended to biologically expressed
proteins, enabling the production of semisynthetic proteins of unlimited size
that contain fluorophores or cross-linking agents at defined positions. Our goal
is to introduce noncoded amino acids and other chemical groups into proteins
to better understand the molecular basis of protein function.
Synthesis of Proteins
Support from the Skaggs Institute has enabled us to improve the methods for
synthesizing proteins. The assembly of large proteins requires repeated ligation,
deprotection, and purification steps that reduce the yield of the synthesis.
We developed an approach for assembling the peptides on a solid support that
enables us to rapidly synthesize proteins from multiple peptides. In addition
to improving yields, solid-phase ligation will enable us to assemble large numbers
of protein analogs in parallel.
A second area of research is the development of new ligation chemistries.
We developed a benzyl scaffold that can be attached to the N-terminus of a peptide
that will promote efficient peptide ligation in aqueous solution (Fig. 1). Future
efforts will focus on improving the rate of ligation and on removing the scaffold
from the peptide after ligation.
Oxidoreductases, a class of highly conserved enzymes that catalyze the reduction
and oxidation of disulfide bonds in proteins, are coupled to the cellular protein-folding
machinery. The oxidoreductases share a common a/ß fold and a common active
site consisting of Cys-Xaa-Yaa-Cys, yet have widely different oxidation potentials.
Thioredoxin (108 amino acids) and glutaredoxin-3 (83 amino acids) are excellent
candidates for analysis via total chemical synthesis. Incorporation of unnatural
amino acids into these proteins will enable us to probe the secondary determinants
of oxidation potential and to gain insights into the mechanism of reduction of
Protein Scaffolds For Enzymatic Catalysis
Many enzymes have a common 3-dimensional fold despite having large differences
in their substrate specificity and mechanism. We are using chemical synthesis
to introduce unnatural structural elements and functional groups into the oxidoreductase
fold in order to design new protein-based catalysts. In a similar study, done
in collaboration with E. Keinan, the Skaggs Institute, and F. Grynszpan, The
Scripps Research Institute, we are using a small hexameric enzyme as a scaffold
to generate novel amine-catalyzed reactions.
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 chemical ligation to cyclize the polypeptide chain. Cyclization of a peptide
may alter the folding properties by causing changes in the amino acid connectivity.
Recently, we 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 3-dimensional topologically
Beligere, G.S., Dawson, P.E. Synthesis of a three zinc finger protein,
Zif268, by native chemical ligation. Biopolymers 51:363, 1999.
Brik, A., Keinan, E., Dawson P.E. Protein synthesis by solid-phase
chemical ligation using a safety catch linker. J. Org. Chem. 65:3829, 2000.
Cho, S., Dawson, P.E., Dawson, G. In vitro depalmitoylation of neurospecific
peptides: Implication for infantile neuronal ceroid lipofuscinosis. J. Neurosci.
Res. 59:32, 2000.
Deniz, A.A., Laurence, T.A., Beligere, G.S., Dahan, M., Martin, A.B.,
Chemla, D.S., Dawson, P.E., Schultz, P.G., Weiss, S. Single-molecule protein
folding: Diffusion fluorescence resonance energy transfer studies of the denaturation
of chymotrypsin inhibitor 2. Proc. Natl. Acad. Sci. U. S. A. 97:5179, 2000.
King, C.C., Zenke, F.T., Dawson, P.E., Dutil, E.M., Newton, A.C., Hemmings,
B.A., Bokoch, G.M. Sphingosine is a novel activator of 3-phosphoinositide-dependent
kinase 1. J. Biol. Chem. 275:10108, 2000.
Marx, P.F., Hackeng, T.M., Dawson, P.E., Griffin, J.H., Meijers, J.C.,
Bouma, B.N. Inactivation of active thrombin-activatable fibrinolysis inhibitor
takes place by a process that involves conformational instability rather than
proteolytic cleavage. J. Biol. Chem. 275:12410, 2000.
Offer, J., Dawson, P.E. Nα-2-Mercaptobenzylamine-assisted chemical
ligation. Org. Lett. 1:23, 2000.