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

Choy Theng Loh, Luke A. Adams, Bim Graham, Gottfried Otting

Research output: Contribution to journalArticleResearchpeer-review

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 54 kDa. The 1H-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 R2 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 T2 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.

Original languageEnglish
Pages (from-to)287-293
Number of pages7
JournalJournal of Biomolecular NMR
Volume71
Issue number4
DOIs
Publication statusPublished - 1 Aug 2018

Cite this

@article{b74f75a3e76d45b6a60ef534a3d51387,
title = "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 54 kDa. The 1H-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 R2 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 T2 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.",
author = "Loh, {Choy Theng} and Adams, {Luke A.} and Bim Graham and Gottfried Otting",
year = "2018",
month = "8",
day = "1",
doi = "10.1007/s10858-017-0157-y",
language = "English",
volume = "71",
pages = "287--293",
journal = "Journal of Biomolecular NMR",
issn = "0925-2738",
publisher = "Springer-Verlag London Ltd.",
number = "4",

}

Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy. / Loh, Choy Theng; Adams, Luke A.; Graham, Bim; Otting, Gottfried.

In: Journal of Biomolecular NMR, Vol. 71, No. 4, 01.08.2018, p. 287-293.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy

AU - Loh, Choy Theng

AU - Adams, Luke A.

AU - Graham, Bim

AU - Otting, Gottfried

PY - 2018/8/1

Y1 - 2018/8/1

N2 - 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 54 kDa. The 1H-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 R2 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 T2 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.

AB - 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 54 kDa. The 1H-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 R2 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 T2 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.

UR - http://www.scopus.com/inward/record.url?scp=85035804853&partnerID=8YFLogxK

U2 - 10.1007/s10858-017-0157-y

DO - 10.1007/s10858-017-0157-y

M3 - Article

VL - 71

SP - 287

EP - 293

JO - Journal of Biomolecular NMR

JF - Journal of Biomolecular NMR

SN - 0925-2738

IS - 4

ER -