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 H-NMR signals of the tert-butyl and TMS groups were compared to the H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54 kDa. The 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 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 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.
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http://dx.doi.org/10.1007/s10858-017-0157-y | DOI Listing |
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