A three-dimensional NMR experiment is presented for correlating NOEs between methyl groups in protonated, 15N,13C-labeled or methyl protonated, highly deuterated 15N,13C-labeled proteins. The high resolution of this experiment facilitates the assignment of NOEs between methyls that can be poorly resolved in other three- and four-dimensional experiments and extends the utility of solution-based NMR structural studies to proteins in the 40 kDa molecular weight regime. Applications to methyl protonated, highly deuterated samples of the 370 residue maltose binding protein (122 methyl groups) and the dimer of the 124 residue amino terminal domain of human STAT-4 (59 methyls) are presented. The method is also well suited for studies of fully protonated proteins, as demonstrated with an application involving the 160 residue phosphotyrosine binding domain from Drosophila Numb. Distance restraints obtained from the present experiment are particularly useful in the generation of global protein folds since many methyls are located in hydrophobic protein cores and inter-methyl restraints therefore link elements of secondary structure that are often distant in the primary sequence.
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