Application Note 16

Specific Isotopic Labeling of Methyl Groups Has Extended the Molecular Weight Limits for NMR Studies of Protein Structure and Dynamics

John B. Jordan¹ and Richard W. Kriwacki^1,2

1. St. Jude Children’s Research Hospital, Department of Structural Biology, Memphis, TN 38105 USA
2. University of Tennessee Health Sciences Center, Department of Molecular Sciences, Memphis, TN 38163 USA

Nuclear magnetic resonance (NMR) spectroscopy has emerged as the preeminent tool in solution studies of protein structure,^1-3 dynamics,^1,4-10 and intermolecular interactions.^11-14 A key limitation, however, was that only relatively small proteins could be studied. Recent advances in isotope labeling,^15-17 the development of TROSY-based methods,¹⁸ and the advent of cryogenic probes¹⁹ have extended the size limit associated with protein NMR. One widely used isotopic-labeling scheme is perdeuteration, which has been shown to mitigate rapid heteronuclear (e.g. ¹³C and ¹⁵N) relaxation.²⁰ A significant drawback to this labeling scheme, however, is that it drastically reduces the number of ¹H-¹H NOEs that can be measured and subsequently used for protein structure determination. To allow the measurement of structurally informative NOEs in perdeuterated proteins, Kay and coworkers demonstrated the use of specific α-keto acid precursors to selectively protonate methyl groups of Ile, Leu, and Val residues^15,16,21,22 (referred to here as ILV). The selective incorporation of ¹H and ¹³C in methyl groups of these residues allowed the measurement of structurally informative NOEs with high sensitivity and resolution.¹ ILV residues are abundant (~21% of all residues)^23,24 and are often found within the hydrophobic cores of globular proteins. These factors facilitate the assignment and measurement of numerous long-range amide-methyl and methyl-methyl NOEs,^16,22,25 which allow global folds of relatively large proteins to be determined. The utility of specific methyl protonation was demonstrated by Tugarinov, et al., through near complete assignment of ILV methyl resonances²⁶ and global fold determination of the 723-residue enzyme, malate synthase G (MSG),²³ and has served to extend the size limitations associated with protein NMR. The following is a brief summary of the ILV methyl group labeling schemes that are available and how these are utilized with current-day protein NMR methods. The reader is referred to the scientific literature for a more complete discussion of these topics.

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