Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy

摘要

The amino acids 4-( tert -butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p -cyano-phenylalanine. The_(1)H-NMR signals of the tert -butyl and TMS groups were compared to the_(1)H-NMR signal of tert -butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54?kDa. The_(1)H-NMR resonance of the TMS group appeared near 0?ppm in a spectral region with few protein resonances, facilitating the observation of signal changes in response to ligand binding. In all proteins, the R ~(2)relaxation rate of the tert -butyl group of Tbf was only little greater than that of Tby (less than two-fold). Deuteration of the phenyl ring of Tbf made only a relatively small difference. The effective T ~(2)relaxation time of the TMS signal was longer than 140?ms even in the 54?kDa system.