The Skaggs Institute
for Chemical Biology

Scientific Report 2007

New Amino Acid Building Blocks

P.G. Schultz, J Grünewald, E.M. Brustad, J.H. Mills, H. Lee, T.S. Young, F. Peters, M. Bushey, S. Schiller

Almost all processes of living cells, from gene regulation and information processing to photosynthesis, are carried out by proteins. These large molecules are synthesized from 20 amino acid building blocks. This set of 20 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 why every form of life has the same set of building blocks. Why not 21 or more? Moreover, if we can add new amino acid building blocks to the genetic code, will we be able to create proteins or even cells 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 bacteria. Using this approach, we effectively expanded the genetic code of both prokaryotes and eukaryotes by genetically encoding new amino acids (including photoaffinity labels, chemically reactive amino acids, posttranslationally modified amino acids, and amino acids with altered electronic and steric properties) in response to unique 3- and 4-base codons.

Currently, we are exploring additional amino acids with novel biological and physicochemical properties, other organisms, and a number of biomedical applications of this technology. For example, in the past year we have (1) genetically encoded a dansyl-containing amino acid, the infrared probe para-cyanophenylalanine, a variety of photocaged amino acids, cysteine homologs, amino acids containing boronate or ferrocene side chains, long-chain alkane-containing amino acids, and sulfotyrosine; (2) expanded this method to mammalian cells by adapting orthogonal tRNA–aminoacyl-tRNA synthetase pairs evolved in yeast; (3) optimized the expression of mutant proteins in Escherichia coli and yeast; and (4) solved structures of benzophenone- and bipyridyl-specific aminoacyl-tRNA synthetases.

In a project to identify and characterize small molecules that can be used in regenerative medicine, we have (1) carried out a cell-based screen and identified molecules that induce the proliferation of beta cells (for the treatment of type 1 diabetes); (2) identified molecules that control Wnt and Hedgehog signaling, 2 key pathways involved in development and cancer; (3) initiated a screen to identify small molecules that upregulate levels of the survival motor neuron 2 protein (ultimately for the treatment of spinal muscular atrophy); and (4) identified molecules that induce epidermal keratinocyte differentiation and molecules that reprogram oligodendrocyte progenitor cells. During the next year, we will characterize the molecular targets and biological activities of these small molecules and attempt to enhance their pharmacologic properties.

Publications

Chen, S., Schultz, P.G., Brock, A. An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae. J. Mol. Biol. 371:112, 2007.

Hong, J., Lee, J., Min, K.-H., Walker, J.R., Peters, E.C., Gray, N.S., Cho, C.Y., Schultz, P.G. Identification and characterization of small-molecule inducers of epidermal keratinocyte differentiation. ACS Chem. Biol. 2:171, 2007.

Lee, J., Wu, X., Pasca di Magliano, M., Peters, E.C., Wang, Y., Hong, J., Hebrok, M., Ding, S., Cho, C.Y., Schultz, P.G. A small-molecule antagonist of the Hedgehog signaling pathway. Chembiochem 8:1916, 2007.

Liu, W., Brock, A., Chen, S., Chen, S., Schultz, P.G. Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nat. Methods 4:239, 2007.

Lyssiotis, C.A., Walker, J.R., Wu, C., Kondo, T., Schultz, P.G., Wu, X. Inhibition of histone deacetylase activity induces developmental plasticity in oligodendrocyte precursor cells. Proc. Natl. Acad. Sci. U. S. A. 104:14982, 2007.

Mukherji, M., Brill, L.M., Ficarro, S.B., Hampton, G.M., Schultz, P.G. A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways. Biochemistry 45:15529, 2006.

Tian, F., Debler, E.W., Millar, D.P., Deniz, A.A., Wilson, I.A., Schultz, P.G. The effects of antibodies on stilbene excited-state energetics. Angew. Chem. Int. Ed. 45:7763, 2006.

Tippmann, E.M., Schultz, P.G. A genetically encoded metallocene containing amino acid. Tetrahedron 63:6182, 2007.

Xie, J., Liu, W., Schultz, P.G. A genetically encoded bidentate, metal-binding amino acid. Angew. Chem. Int. Ed. 46:9239, 2007.

Xie, J., Supekova, L., Schultz, P.G. A genetically encoded metabolically stable analogue of phosphotyrosine in Escherichia coli. ACS Chem. Biol. 2:474, 2007.

Zhang, Q., Major, M., Takanashi, S., Camp, N., Nishiya, N., Ginsberg, M., Jian, X., Randasso, P.A., Schultz, P.G., Moon, R.T., Ding, S. Small-molecule synergist of the Wnt/β-catenin signaling pathway. Proc. Natl. Acad. Sci. U. S. A. 104:7444, 2007.