Site-Specific Incorporation of 7-Fluoro-L-tryptophan into Proteins by Genetic Encoding to Monitor Ligand Binding by ¹⁹F NMR Spectroscopy

Abstract

A mutant aminoacyl-tRNA synthetase identified by a library selection system affords site-specific incorporation of 7-fluoro-L-tryptophan in response to an amber stop codon. The enzyme allows the production of proteins with a single hydrogen atom replaced by a fluorine atom as a sensitive nuclear magnetic resonance (NMR) probe. The substitution of a single hydrogen atom by another element that is as closely similar in size and hydrophobicity as possible minimizes possible perturbations in the structure, stability, and solubility of the protein. The fluorine atom enables site-selective monitoring of the protein response to ligand binding by 19 F NMR spectroscopy, as demonstrated with the Zika virus NS2B-NS3 protease. KEYWORDS: fluoro-tryptophan, 19 F NMR spectroscopy, ligand binding, genetic encoding, noncanonical amino acids, pyrrolysyl-tRNA synthetase 19 F NMR spectroscopy of proteins labeled with fluorine atoms is becoming increasingly popular for studying protein structure, conformational changes, and protein−ligand interactions. 1−3 19

Synthesis of fluorinated leucines, valines and alanines for use in protein NMR

“…35 The apparent resilience of the fold of GB1 towards global substitution of canonical amino acids for fluorinated analogues may be attributed to the relatively small number (three each) of leucine and valine residues that are buried or partially buried in the hydrophobic core. Work is in progress to find FAA-specific aminoacyl–tRNAs that enable the incorporation of a single FAA, 36 to minimize sample heterogeneity and detect specific fluorine–fluorine contacts. 37…”

Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids

“…For example, the genetic encoding systems recently established for p-SF 5 -phenylalanine and 7fluoro-tryptophan delivered 19 F NMR line widths approaching 100 Hz and more for relatively small proteins of molecular weights below 26 kDa. 21,22 Owing to the flexibility of their side chains, the amino acid probes of the present work compare very favorably with other, less flexible, non-canonical amino acids containing a TMS group. Furthermore, the 1 H chemical shift of the TMS group is in a more favorable spectral region when it is linked to a nonaromatic moiety.…”

“…The availability of a genetic encoding system, which allows the facile incorporation of an NMR probe at a single site, presents an important advance, but enhanced flexibility of the NMR probe with respect to the core of the protein remains critically important to limit the line widths observed in high molecular weight systems. For example, the genetic encoding systems recently established for p -SF 5 -phenylalanine and 7-fluoro-tryptophan delivered 19 F NMR line widths approaching 100 Hz and more for relatively small proteins of molecular weights below 26 kDa. , …”

“…Aminoacyl-tRNA synthetases (RS) specific for TMSNK were selected from a library of RS mutants based on the pyrrolysyl-tRNA synthetase (PylRS) from the methanogenic archaeon ISO4-G1, using fluorescence-activated cell sorting (FACS) as described previously. ,, To carry out the selection, the library plasmid pBK-G1RS was transformed into Escherichia coli DH10B cells harboring the selection plasmid pBAD-H6RFP . Following recovery from transformation, the culture was directly inoculated into a flask with 25 mL LB medium containing 100 mg/L carbenicillin and 50 mg/L kanamycin and supplied with 0.4% l -arabinose and 2 mM TMSNK, which served as the sample for the first round of positive selection (1P+).…”

Site‐directed mutagenesis improves the practical application of L‐glutamic acid decarboxylase inEscherichia coli

“…Such a mutation changes the structure and orientation of Gad B under neutral conditions [36,37], and the contact effect is weakened (as shown in Figure 4B). Second, the R group of the amino acid residues that form the βlamellar must not be too large, so stretching is conducive to the chain, under the condition of the peptide chain extension, most amino acids hydrophilic interaction with the surrounding water, a large number of hydrophilic amino acid residues of side chain groups and melting by solvent, which contribute to the formation of hydrophobic internal role, hydrophobic packaging to form active center cavities is also a major determinant of protein stability [38]. Guo et al [39], using online prediction software PoPMiSiC, to predict the change of free energy of single point mutation of Bacillus subtilis chitosanase Bs Csn46A, the specific activity of the three mutants increased 1.69, 1.97, and 2.15 times, respectively.…”