The use of selective deuteration for the sequence specific ¹H NMR assignment of larger proteins

Abstract

The possibility of extending NMR methods for structure determination to larger proteins (MW > 10 kD) depends on the development of isotopic labeling protocols for the simplification of their NMR spectra (isotopic spectral editing). We describe here the successful use of selective deuteration to obtain sequence specific assignments for (thus far) more than 50% of the residues of the trp repressor protein (25 kD). This is the largest protein for which detailed sequence specific assignments have been attempted to‐date.

THE USE OF2H,13C,15N MULTIDIMENSIONAL NMR GTO STUDY THE STRUCTURE AND DYNAMICS OF PROTEINS

“…In particular, substitution of aliphatic/aromatic protons with deuterons results in impressive sensitivity gains in NOESY spectra that record NH-NH correlations (94,156). Initially demonstrated in perdeuterated systems, similar benefits have also been observed for proteins and peptides that are protonated at specific positions in an otherwise highly deuterated background (5,124,132,158). The sensitivity gains in these NOESY data sets are largely the result of reduced NH linewidths, as described above.…”

“…In the case of highly deuterated proteins containing protonated amino acids, NOE spectra have been recorded with long mixing times specifically to promote intra-residue spin diffusion. In this way sidechain protons for several dimeric proteins with molecular weights above 20 kDa have been assigned (4,5,133).…”