In vivo Incorporation of Unnatural Amino Acids

Scripps Research Institute, The, La Jolla CA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): In the previous funding period we developed a general method that allows one to systematically add amino acids with novel physical, chemical and biological properties to the genetic code of E. coil and yeast. These amino acids are genetically encoded efficiently and with high fidelity by the amber codon TAG and quadruplet codon AGGA. In this proposal we will extend this work in several directions: (1) We will solve the three dimensional crystal structures of several E. coil and yeast orthogonal synthetases that encode unnatural amino acids to better understand how to evolve synthetase selectivity; (2) We will genetically encode a number of N- and O-linked monosaccharides in E. coil and yeast and demonstrate that they can be used to generate selectively glycosylated proteins; (3) We will develop a robust and general method for generating tRNA - synthetase pairs that can insert unnatural amino acids in mammalian cells; (4) We will genetically encode a number of biophysical probes in E. coil and yeast (including fluorescent amino acids, spin-labeled amino acids, photocaged amino acids, heavy atoms containing amino acids, and amino acids that can be used as selective NMR and IR probes). These studies should dramatically expand our ability to manipulate protein structure in order to create proteins with new functions and to probe protein structure and function in vitro and in vivo. [unreadable] [unreadable]