tailieunhanh - Báo cáo khoa học: Charging of tRNA with non-natural amino acids at high pressure

We show a simple and reliable method of tRNA aminoacylation with natural, as well as non-natural, amino acids at high pressure. Such specific and noncognate tRNAs can be used as valuable substrates for protein engineering. Aminoacylation yield at high pressure depends on the chem-ical nature of the amino acid used and it is up to 10%. | iFEBS Journal Charging of tRNA with non-natural amino acids at high pressure Malgorzata Giel-Pietraszuk and Jan Barciszewski Institute of Bioorganic Chemistry Polish Academy of Sciences Poznan Poland Keywords high pressure non-naturalamino acids tRNA charging Correspondence Jan Barciszewski Institute of Bioorganic Chemistry Polish Academy of Sciences Noskowskiego 12 14 61-704 Poznan Poland Fax 49 61 852 05 32 Tel 48 61 852 85 03 ext. 132 E-mail Received 3 October 2005 revised 25 April 2006 accepted 9 May 2006 doi We show a simple and reliable method of tRNA aminoacylation with natural as well as non-natural amino acids at high pressure. Such specific and noncognate tRNAs can be used as valuable substrates for protein engineering. Aminoacylation yield at high pressure depends on the chemical nature of the amino acid used and it is up to 10 . Using CoA which carries two potentially reactive groups -SH and -OH as a model compound we showed that at high pressure amino acid is bound preferentially to the hydroxyl group of the terminal ribose ring. Site-specific incorporation of non-natural amino acids into proteins is an increasingly emerging field because of the application of non-natural amino acids as biophysical probes in structure-function studies. Moreover modified peptides may be key pharmaceuticals for the treatment of a variety of diseases 1 . Among these compounds are protease inhibitors a classic example of which are the HIV protease inhibitors 2 3 . Replacement of methionine with selenomethionine has been used extensively for phase determination in protein crystallography and the exchange of 4-fluorotryptophan for tryptophan has been used in NMR analysis. Studies on the function and properties of proteins require mutants containing amino acid analogues for example thioproline at multiple sites that do not influence protein function including immunogenicity but may serve as promising vehicles .