In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance Spectroscopy

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• 作者：Susan E. Cellitti ; David H. Jones ; Leanna Lagpacan ; Xueshi Hao ; Qiong Zhang ; Huiyong Hu ; Scott M. Brittain ; Achim Brinker ; Jeremy Caldwell ; Badry Bursulaya ; Glen Spraggon ; Ansgar Brock ; Youngha Ryu ; T
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：July 23, 2008
• 年：2008
• 卷：130
• 期：29
• 页码：9268 - 9281
• 全文大小：3273K
• 年卷期：v.130,no.29(July 23, 2008)
• ISSN：1520-5126

文摘

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl−tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF₃Phe), ¹³C/¹⁵N-labeled p-methoxyphenylalanine (OMePhe), and ¹⁵N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF₃Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in ¹H−¹⁵N HSQC, ¹H−¹³C HSQC, and ¹⁹F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF₃Phe mutants of an active site tyrosine inhibited binding; incorporating ¹⁵N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.