The binding of tetrameric E. coli single‐stranded DNA‐binding protein (SSB) to single‐stranded DNA (ssDNA) was investigated using genetically encoded noncanonical amino acids (ncAA) as site‐specific probes for detection by nuclear magnetic resonance (NMR) spectroscopy. Under the conditions used (300 m m NaCl, pH 7.2), the NMR spectra confirmed the equivalence of the monomeric subunits in the absence of ssDNA. Most of the probes responded to the binding of ssDNA by changes in chemical shifts and line width and distinguished between the presence of segments of cytidine versus thymidine. Although ssDNA‐binding breaks the fourfold symmetry of the SSB tetramer, the probes sensed closely similar chemical environments in all four monomeric subunits. By comparing the performance of twelve different NMR‐active ncAAs, this work identified N 6 ‐trifluoroacetyl‐L‐lysine (TFAK) as the ncAA sensing different ssDNAs with the best spectral resolution. In addition, we report aminoacyl‐tRNA synthetases for the genetic encoding of 3,5‐difluoro‐L‐tyrosine (3,5‐diFTyr), 2,6‐difluoro‐L‐tyrosine (2,6‐diFTyr), and m CF 3 ‐phenylalanine. The SSB construct was sensitive to precipitation under NMR conditions. The fluorinated ncAAs altered the rates of precipitation which varied even between fluorotryptophan isomers installed at a barely solvent‐accessible site. Nonetheless, the NMR‐active ncAAs proved suitable for probing a marginally stable protein system of ca. 100 kDa molecular weight without isotope labelling and at low concentration. The current data suggest that 19 F spins attached to flexible solvent‐exposed amino acid side chains guard better against protein precipitation than fluorinated aromatic amino acids despite the latter being more attractive for their close structural similarity to their canonical amino acid counterparts.
Tharayil et al. (Tue,) studied this question.