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 co-expressing 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 3 Phe), 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 3 Phe. 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 E. 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, 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 3 Phe 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.

Second-generation sulfonamide inhibitors of d-glutamic acid-adding enzyme: Activity optimisation with conformationally rigid analogues of d-glutamic acid

“…The results were determined as residual activities (RAs) of the enzyme in the presence of 500 mM or 1 mM of each compound, and also as IC 50 and K i values for the most active compounds ( Table 2). …”

“…As expected, slightly better inhibition was seen with compounds 31f-31i, which have two polar groups at various positions on the aromatic ring. The most interesting data was from the comparison of the inhibitory potencies of compounds 31f (RA at 500 mM, 76%) and 31i (RA at 1 mM, 8%; IC 50 ¼ 127 mM). The importance of the carboxyl group at the para position with respect to the sulfonamide moiety was evident, in comparison with the hydroxyl group.…”

“…1) was developed, which showed IC 50 values in the low micromolar range [26]. Peptide inhibitors of MurD from Pseudomonas aeruginosa were obtained by screening of phage display libraries using competitive biopanning approaches, with the best IC 50 value obtained of 4 mM [27]. Pyrazole derivatives [28] and pulvinones [29] have been reported to be potential inhibitors of MurBeMurD and MurAeMurD, respectively.…”

“…Recently, the high-resolution co-crystal structures of MurD with N-sulfonyl-L-Glu (9) (IC 50 ¼ 710 mM) and N-sulfonyl-D-Glu (10) (IC 50 ¼ 280 mM) (Fig. 1) were reported.…”

O-tert-Butyltyrosine, an NMR Tag for High-Molecular-Weight Systems and Measurements of Submicromolar Ligand Binding Affinities

“…For example, Geierstanger and co-workers have shown that the NMR resonances of the methyl-and CF 3 -groups of pmethoxyphenylalanine (OMePhe) and p-trifluoromethoxyphenylalanine (OCF 3 Phe) can readily be observed after sitespecific incorporation at different locations of a 33 kDa protein. 9 The signals of the newly introduced 13 CH 3 -and CF 3 groups were readily observed in two-dimensional (2D) 13 C HSQC and 1D 19 F NMR spectra, respectively. Similarly, Mehl and co-workers demonstrated that site-specific incorporation of trifluoromethylphenylalanine (CF 3 Phe) produced 1D 19 F NMR spectra that allowed distinction of different ligand-and metalbound states in 48 and 98 kDa homodimers.…”