1H-15N heteronuclear NMR studies of Escherichia coli thioredoxin in samples isotopically labeled by residue type

Abstract

Ten samples of Escherichia coli thioredoxin were individually isotopically enriched by residue type via growth of an appropriate auxotrophic strain on media supplemented with one 2H, 15N-enriched amino acid. 1H observe-heteronuclear decoupling experiments were conducted on these samples making use of the 95-Hz 1H-15N amide J1 coupling. Subtraction of near-resonance from off-resonance 15N decoupled spectra generated difference patterns corresponding only to protons directly bonded to 15N nuclei. For the ten different enriched residue types observed to date, every labeled position (60) has been observed as a resolved resonance. The spectral dispersion in both the 1H and the 15N dimensions was roughly 1500 Hz (at 500-MHz field strength) with rather little apparent dependence on residue type. With the exception of the glycine-enriched sample, the range of the J1 coupling constants was not much greater than the precision of the measurements (1.5-2.0 Hz). However, for the glycine residues the J1 amide coupling values varied over a range of 10 Hz.