Determination of all nOes in ¹H-¹³ C-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 ¹H-¹³C-ILV methyl-labelled proteins that are otherwise uniformly deuterated and ¹⁵N 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 ¹⁵N and ¹³C 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 (¹H,¹³ C)-ILV-(¹H)-FY-(U-²H,¹⁵N) sample of a 37 kDa di-domain of the E. coli enterobactin synthetase module EntF.