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The Skaggs Institute
for Chemical Biology


Scientific Report 2008




New Amino Acid Building Blocks

P.G. Schultz, E.M. Brustad, J. Grünewald, J. Guo, H.S. Lee, C.C. Liu, F.B. Peters, T.S. Young

Almost all processes of living cells, from gene regulation and information processing to photosynthesis, are carried out by proteins. These are large molecules 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 rather interesting questions: Why does every form of life have 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 continue to address this issue by using 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 showed that tolerance against the self-antigen TNF-α in mice can be broken by the selective introduction of p-nitrophenylalanine. This research promises to greatly facilitate the generation of cancer vaccines and vaccines against third-world pathogens; we are currently extending it to lung and breast cancer vaccines and vaccines against tuberculosis, malaria. and HIV disease. We also genetically encoded a boronate-containing amino acid for the scarless purification of proteins, selective protein modification, and the development of selective antibodies to both glycoproteins and serine proteases involved in viral infections and cancer. In addition, we genetically encoded a hydroxyquinoline metal ion–binding amino acid for heavy-atom phasing in protein structure determination and for the introduction of radioisotopes into antibodies for cancer therapy and imaging.

In other studies, we used a phenylselenide-containing amino acid to generate proteins containing posttranslationally modified lysine residues to study the role of histone modification in the epigenetics of cancer and developmental biology. We also showed that phage display can be used to select anti-gp120 sulfotyrosine-containing antibodies with enhanced affinities relative to the naturally sulfated anti-gp120 antibody. This result shows for the first time that an expanded genetic code can confer an evolutionary advantage and may lead to the generation of therapeutic proteins or peptides with improved biological or pharmacologic properties due to the presence of unnatural amino acids.

We also genetically encoded the fluorophore prodan in mammalian cells to facilitate cellular studies of protein structure, function, and localization; genetically encoded an unnatural amino acid that can cleave the polypeptide backbone in a light-dependent fashion as a cell biological tool; and biosynthesized DNA-binding proteins that can oxidatively cleave the DNA backbone in a sequence-specific fashion as a probe of protein-DNA recognition. Furthermore, we showed that unnatural amino acids can be used to selectively introduce fluorescence resonance energy transfer pairs into proteins for single-molecule imaging studies, and we developed a high-yield Pichia expression system to produce therapeutic proteins containing unnatural amino acids.

Publications

Brustad, E., Bushey, M.L., Brock, A., Chittuluru, J., Schultz, P.G. A promiscuous aminoacyl-tRNA synthetase that incorporates cysteine, methionine, and alanine homologs into proteins. Bioorg. Med. Chem. Lett. 18:6004, 2008.

Brustad, E., Bushey, M.L., Lee, J.W., Groff, D., Liu, W., Schultz, P.G. A genetically encoded boronate-containing amino acid. Angew. Chem. Int. Ed. 47:8220, 2008.

Grünewald, J., Tsao, M.-L., Perera, R., Dong, L., Niessen, F., Wen, B.G., Kubitz, D.M., Smider, V.V., Ruf, W., Nasoff, M., Lerner, R.A., Schultz, P.G. Immunochemical termination of self-tolerance. Proc. Natl. Acad. Sci. U. S. A. 105:11276, 2008.

Guo, J., Wang, J., Anderson, J.C., Schultz, P.G. Addition of an α-hydroxy acid to the genetic code of bacteria. Angew. Chem. Int. Ed. 47:722, 2008.

Guo, J., Wang, J., Lee, J.-S., Schultz, P.G. Site-specific incorporation of methyl- and acetyl-lysine analogues into recombinant proteins. Angew. Chem. Int. Ed. 47:6399, 2008.

Lee, J., Hong, J., Nam, T.-G., Peters, E.C., Orth, A.P., Geierstanger, B.H., Goldfinger, L.E., Ginsberg, M.H., Cho, C.Y., Schultz, P.G. A small molecule inhibitor of α4 integrin-dependent cell migration. Bioorg. Med. Chem., in press.

Lemke, E.A., Summerer, D., Geierstanger, B.H., Brittain, S.M., Schultz, P.G. Control of protein phosphorylation with a genetically encoded photocaged amino acid. Nat. Chem. Biol. 3:769, 2007.

Tippmann, E.M., Liu, W., Summerer, D., Mack, A.V., Schultz, P.G. A genetically encoded diazirine photocrosslinker in Escherichia coli. ChemBioChem 8:2210, 2007.

 

Peter Schultz, Ph.D.
Scripps Family Chair Professor

Schultz Web Site