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-   -   Genetically encoded amino acids with tert -butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy (http://www.bionmr.com/forum/journal-club-9/genetically-encoded-amino-acids-tert-butyl-trimethylsilyl-groups-site-selective-studies-proteins-nmr-spectroscopy-25378/)

nmrlearner 12-02-2017 02:54 PM

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

Abstract

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 1H-NMR signals of the tert-butyl and TMS groups were compared to the 1H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54Â*kDa. The 1H-NMR resonance of the TMS group appeared near 0Â*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 140Â*ms even in the 54Â*kDa system.



Source: Journal of Biomolecular NMR


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