title: A Semiautomated Assignment Protocol for Methyl Group Side Chains in Large Proteins.
abstract: The developments of biosynthetic specific labeling strategies for side-chain methyl groups have allowed structural and dynamic characterization of very large proteins and protein complexes. However, the assignment of the methyl-group resonances remains an Achilles' heel for NMR, as the experiments designed to correlate side chains to the protein backbone become rather insensitive with the increase of the transverse relaxation rates. In this chapter, we outline a semiempirical approach to assign the resonances of methyl-group side chains in large proteins. This method requires a crystal structure or an NMR ensemble of conformers as an input, together with NMR data sets such as nuclear Overhauser effects (NOEs) and paramagnetic relaxation enhancements (PREs), to be implemented in a computational protocol that provides a probabilistic assignment of methyl-group resonances. As an example, we report the protocol used in our laboratory to assign the side chains of the 42-kDa catalytic subunit of the cAMP-dependent protein kinase A. Although we emphasize the labeling of isoleucine, leucine, and valine residues, this method is applicable to other methyl group side chains such as those of alanine, methionine, and threonine, as well as reductively methylated cysteine side chains.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Jonggul
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA. Electronic address: vegli001@umn.edu.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Veglia
mesh_terms: D020134:Catalytic Domain; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins
keywords: Auto-assignment of methyl groups; Large systems; Methyl labeling; Protein NMR; Protein kinase A; Side-chain assignment