The ability to expand the genetic code to encode chemical entities beyond the 20 amino acids found in nature requires new and clever methods of cellular engineering. In this proposal, we plan to reprogram the existing rules of translation to incorporate photoreactive analogues. We provide an in vivo approach towards the site-specific incorporation of photoreactive analogues and attempt to utilize the wobble pair codon as a secondary coding scheme.
In Specific Aim 1, we propose to expand the ability of the translational machinery to accommodate analogues bearing photoreactive handles using an in vivo selection approach.
In Specific Aim 2, we devise an orthogonal tRNA/aminoacyl-tRNA synthetase (AARS) pair that recognizes the wobble pair codon of phenylalanine and inserts a photoreactive analogue at the designated site. Aside from providing a novel route for the site-specific incorporation of chemical handles, this strategy outlines a general scheme that will enable the incorporation of multiple analogues into a single protein. This technique will prove useful for the study of natural protein structure and function as well as the design of novel semi-synthetic protein architectures.
| Montclare, Jin Kim; Son, Soojin; Clark, Ginevra A et al. (2009) Biosynthesis and stability of coiled-coil peptides containing (2S,4R)-5,5,5-trifluoroleucine and (2S,4S)-5,5,5-trifluoroleucine. Chembiochem 10:84-6 |
| Voloshchuk, Natalya; Lee, Man Xia; Zhu, Wan Wen et al. (2007) Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: improved thermostability by a single-isoleucine mutant. Bioorg Med Chem Lett 17:5907-11 |
| Montclare, Jin Kim; Tirrell, David A (2006) Evolving proteins of novel composition. Angew Chem Int Ed Engl 45:4518-21 |
| Panchenko, Tatyana; Zhu, Wan Wen; Montclare, Jin Kim (2006) Influence of global fluorination on chloramphenicol acetyltransferase activity and stability. Biotechnol Bioeng 94:921-30 |
