The Skaggs Institute
for Chemical Biology

New Amino Acid Building Blocks

P.G. Schultz, E. Brustad, J. Grbic, W. Hur, H. Lee, C. Liu, J. Liu, J. Melnick, J. Mills, K.H. Min, M. Mukherji, R. Perera, E. Tippmann, M. Tsao, J. Xie, Q. Zeng

Almost all processes of living cells, from gene regulation and information processing to photosynthesis, are carried out by proteins. The 20 amino acids used as building blocks in the synthesis of proteins are connected in different combinations to give polymeric structures consisting of anywhere from tens to thousands of amino acids. What is amazing is that every form of life on Earth uses the same set of 20 amino acids to make all proteins. Indeed, this set of amino acids is the basis for the genetic code, the code that specifies the relationship between the nucleotide sequence of a gene and the amino acid sequence of a protein. This fact leads to the rather interesting question of whether 20 amino acids are the optimal number of building blocks for living organisms. If we can add new amino acid building blocks to the genetic code, will we be able to create proteins or even whole organisms with enhanced chemical, physical, or biological properties?

We are addressing this issue by using a number of chemical and molecular biological methods to add new components to the protein biosynthetic machinery of prokaryotic and eukaryotic organisms. This machinery consists of the ribosome, which binds mRNA (a short-lived single-stranded copy of the DNA that encodes a protein) and translates it into a protein sequence. The translation is accomplished by an adapter molecule called tRNA. The genetic code is enforced by enzymes (tRNA aminoacyl synthetases) that load each tRNA with 1 of the common 20 amino acids specified by the genetic code. By adding new components to this biosynthetic machinery, we showed that we can effectively expand the genetic code of Escherichia coli and yeast by adding new amino acids with photoreactive and chemically reactive side chains; glycosylated, fluorescent, and metal-binding amino acids; and amino acids containing heavy atoms and other “nonencoded” functional groups. In addition, we made the first synthetic autonomous organism with a 21 amino acid genetic code and are exploring its ability to evolve in response to a variety of environmental stresses.

In the past year, we solved the crystal structures of a number of these mutant synthetases, which show a high degree of active-site plasticity; developed a phage display system for peptides containing unnatural amino acids; incorporated a fluorescent coumarin amino acid; showed that an azide-containing amino acid can be used to selectively modify proteins via a Staudinger ligation; incorporated thioesters into amino acids to allow the formation of cyclic peptides/proteins; and showed that the codon redundancy of essential E coli genes can be reduced to 41 unique codons.

Our goals for 2006 are to incorporate unnatural amino acids into the multicellular organism Caenorhabditis elegans; optimize incorporation of unnatural amino acids in mammalian cells; incorporate amino acids containing N-acetyl- and N-methyl-lysine as well as amino acids containing nitrile and ferrocene and a series of cysteine homologs; show that unnatural amino acids can be used to make bispecific Fabs and covalent antigen-antibody complexes; incorporate α-hydroxy acids and N-methyl amino acids into proteins; and continue efforts to generate a “reduced degeneracy” E coli genome.

In a separate project, we have been developing and using genomics tools to identify molecules that regulate developmental pathways. Two such pathways are the Wnt and hedgehog signaling pathways, which play critical roles in development and have been implicated in a number of cancers. We have carried out cellular pathway-based reporter screens of combinatorial libraries of small molecules and arrayed cDNAs and short interfering RNAs to find new effectors of this pathway, as well as new gene products involved in this signaling network. To date, we have identified potent small-molecule agonists and antagonists of these pathways as well as novel genes that regulate Wnt signaling. We are characterizing these and other molecules/genes that were identified in these screens. In addition, we have shown that Wnt modulators can play a role in inducing neurogenesis and differentiation. In the next year, we will attempt to characterize the activities of these molecules in cellular and animal models of neurogenesis and cancer.

Publications

Bose, M., Groff, D., Xie, J., Brustad, E., Schultz, P.G. The incorporation of a photoisomerizable amino acid into proteins in E coli. J. Am. Chem. Soc. 128:388, 2006.

Deiters, A., Schultz, P.G. In vivo incorporation of an alkyne into proteins in Escherichia coli. Bioorg. Med. Chem. Lett. 15:1521, 2005.

Ding, S., Schultz, P.G. Small molecules and future regenerative medicine. Curr. Top. Med. Chem. 5:383, 2005.

Liu, J., Bang, A., Kintner, C., Orth, T., Chanda, S., Ding, S., Schultz, P.G. Identification of the Wnt signaling activator leucine-rich repeat in Flightless interaction protein 2 by a genome-wide functional analysis. Proc. Natl.. Acad. Sci. U. S. A. 102:1927, 2005.

Liu, J., Wu, X., Mitchell, B., Kintner, C., Ding, S., Schultz, P.G. A small-molecule agonist of the Wnt signaling pathway. Angew. Chem. Intl. Ed. 44:1987, 2005.

Tsao, M., Tian, F., Schultz, P.G. Selective Staudinger modification of proteins containing p-azidophenylalanine. Chembiochem 6:2147, 2005.

Turner, J.M., Graziano, J., Spraggon, G., Schultz, P.G. Structural characterization of a p-acetylphenylalanyl aminoacyl-tRNA synthetase. J. Am. Chem. Soc. 127:14976, 2005.

Xie, J., Schultz, P.G. Adding amino acids to the genetic repertoire. Curr. Opin Chem. Biol. 9:548, 2005.