Determination of all nOes in 1H-13C-Me-ILV-U-2H-15N proteins with two time-shared experiments

We present two time-shared experiments that enable the characterization of all nOes in H-1-C-13-ILV methyl-labelled proteins that are otherwise uniformly deuterated and N-15 enriched and possibly selectively protonated for distinct residue types. A 3D experiment simultaneously provides the spectra of a 3D NOESY-HN-TROSY and of a 3D NOESY-HC-PEP-HSQC. Thus, nOes from any protons to methyl or amide protons are dispersed with respect to N-15 and C-13 chemical shifts, respectively. The single 4D experiment presented here yields simultaneously the four 4D experiments HC-HSQC-NOESY-HC-PEP-HSQC, HC-HSQC-NOESY-HN-TROSY, HN-HSQC-NOESY-HN-TROSY and HN-HSQC-NOESY-HC-PEP-HSQC. This allows for the unambiguous determination of all nOes involving amide and methyl protons. The method was applied to a (H-1,C-13)-ILV-(H-1)-FY-(U-H-2,N-15) sample of a 37 kDa di-domain of the E. coli enterobactin synthetase module EntF.