High-Accuracy Distance Measurement between Remote Methyls in Specifically Protonated Proteins

1 H- 1 H distance restraints, derived from nuclear Overhauser effects (NOEs) in macromolecules, are generally restricted to protons close in space and characterized by large inaccuracies due to spin diffusion. Perdeuteration of molecules with selective incorporation of protons into few specific methyl sites greatly reduces the effect of spin diffusion. The approach is demonstrated for the measurement of Ile-δ1 methyl−methyl interactions in the proteins γS-crystallin and ubiquitin and permits observation of the direct NOE transfer between protons up to 12 Å apart. Quantitative evaluation of intermethyl distances from experimental NOEs, in both ubiquitin and γS-crystallin, shows excellent agreement with their solution structure. This method is applicable to large proteins and is expected to be of particular interest for the structure determination of elongated or modular macromolecules or complexes.