unnatural aa

unnatural aa - PERSPECTIVES I N N O VAT I O N A chemical...

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Although the genetic codes of all known organisms specify the same 20 amino acids (with the rare exceptions of selenocysteine 1 and pyrrolysine 2 ), it is clear that numerous proteins require many cofactors and post- translational modifications to carry out their natural functions. Therefore, although a 20-amino-acid code might be sufficient for life, it might not be optimal. Consequently, the development of a method that allows us to encode extra amino acids genetically might facilitate the evolution of proteins, or even entire organisms, with new or enhanced properties. Moreover, the ability to incorporate amino acids with defined steric and electronic properties at unique sites in proteins will provide powerful new tools for exploring protein structure and function in vitro and in vivo . Here, we describe an approach that makes it possible, for the first time, to add new amino acids to the genetic codes of both prokaryotic and eukaryotic organisms. Over 30 unnatural amino acids — including those containing spectroscopic probes, post- translational modifications, metal chelators, photoaffinity labels and other chemical moieties — have been selectively incorpo- rated into proteins with high fidelity and efficiency in response to unique three and four base codons. Methodology General considerations. The incorporation of an unnatural amino acid at a defined site in a protein, directly in a living organism, requires a unique transfer-RNA:codon pair, a cor- responding aminoacyl–tRNA synthetase and significant intracellular levels of the unnatural amino acid 3 . To ensure that the unnatural amino acid is incorporated uniquely at the site specified by its codon, the tRNA must be constructed such that it is not recognized by the endogenous aminoacyl–tRNA synthetases of the host, but functions efficiently in translation (an orthogonal tRNA). Moreover, this tRNA must deliver the novel amino acid in response to a unique codon that does not encode any of the common 20 amino acids. Another requirement for high fidelity is that the cognate aminoacyl–tRNA synthetase (an orthogonal synthetase) aminoacylates the orthogonal tRNA, but does not aminoacylate any of the endogenous tRNAs. Furthermore, this synthetase must aminoacylate the tRNA with only the desired unnatural amino acid and not with the endogenous amino acids. Similarly, the unnatural amino acid cannot be a substrate for the endogenous synthetases if it is to be incorporated uniquely in response to its cognate codon. Last, the unnatural amino acid must be efficiently transported into the cytoplasm when it is added to the growth medium or biosynthesized by the host, and it must be stable in the presence of endogenous metabolic enzymes. Several biochemical methods have pre- viously been developed to insert unnatural amino acids into proteins. However, they require either in vitro protein synthesis or the stoichiometric use of chemically amino- acylated tRNAs 4–6 (which results in low protein yields), or they result in the substitu-
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This note was uploaded on 08/24/2010 for the course BIS 101 taught by Professor Simonchan during the Fall '08 term at UC Davis.

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unnatural aa - PERSPECTIVES I N N O VAT I O N A chemical...

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