title: In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy.
abstract: 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 (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-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 OCF 3Phe. 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 (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F 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 OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-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.
authors.affiliation: Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121-1125, USA.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Cellitti
authors.forename: David H
authors.initials: DH
authors.lastname: Jones
authors.forename: Leanna
authors.initials: L
authors.lastname: Lagpacan
authors.forename: Xueshi
authors.initials: X
authors.lastname: Hao
authors.forename: Qiong
authors.initials: Q
authors.lastname: Zhang
authors.forename: Huiyong
authors.initials: H
authors.lastname: Hu
authors.forename: Scott M
authors.initials: SM
authors.lastname: Brittain
authors.forename: Achim
authors.initials: A
authors.lastname: Brinker
authors.forename: Jeremy
authors.initials: J
authors.lastname: Caldwell
authors.forename: Badry
authors.initials: B
authors.lastname: Bursulaya
authors.forename: Glen
authors.initials: G
authors.lastname: Spraggon
authors.forename: Ansgar
authors.initials: A
authors.lastname: Brock
authors.forename: Youngha
authors.initials: Y
authors.lastname: Ryu
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Uno
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Geierstanger
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010649:Phenylalanine / Q000031:analogs & derivatives*; D010666:Phenylpropionates / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010666:Phenylpropionates; D011506:Proteins; D014443:Tyrosine; D010649:Phenylalanine; D064429:Fatty Acid Synthases; D000604:Amino Acyl-tRNA Synthetases