A Semiautomated Assignment Protocol for Methyl Group Side Chains in Large Proteins

Jonggul Kim, Yingjie Wang, Geoffrey Li, Gianluigi Veglia

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages35-57
Number of pages23
DOIs
StatePublished - 2016

Publication series

NameMethods in Enzymology
Volume566
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
This work was supported in part by the NIH (GM100310 and GM72701 to G.V. and T32 AR007612 to J.K.). NMR experiments were carried out at the Minnesota NMR Center and FLAMEnGO 2.0 calculations at the Minnesota Supercomputing Institute.

Keywords

  • Auto-assignment of methyl groups
  • Large systems
  • Methyl labeling
  • Protein NMR
  • Protein kinase A
  • Side-chain assignment

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