Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics

Igor Dikiy, Lindsay D. Clark, Kevin H Gardner, Daniel M Rosenbaum

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

Abstract

Membrane proteins, and especially G-protein coupled receptors (GPCRs), are increasingly important targets of structural biology studies due to their involvement in many biomedically critical pathways in humans. These proteins are often highly dynamic and thus benefit from studies by NMR spectroscopy in parallel with complementary crystallographic and cryo-EM analyses. However, such studies are often complicated by a range of practical concerns, including challenges in preparing suitably isotopically labeled membrane protein samples, large sizes of protein/detergent or protein/lipid complexes, and limitations on sample concentrations and stabilities. Here we describe our approach to addressing these challenges via the use of simple eukaryotic expression systems and modified NMR experiments, using the human adenosine A2A receptor as an example. Protocols are provided for the preparation of U-2H (13C,1H-Ile δ1)-labeled membrane proteins from overexpression in the methylotrophic yeast Pichia pastoris, as well as techniques for studying the fast ns–ps sidechain dynamics of the methyl groups of such samples. We believe that, with the proper optimization, these protocols should be generalizable to other GPCRs and human membrane proteins.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsA. Joshua Wand
PublisherAcademic Press Inc.
Pages37-65
Number of pages29
ISBN (Print)9780128138601
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Enzymology
Volume614
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Dynamics
  • GPCRs
  • Isotopic labeling
  • Membrane proteins
  • NMR spectroscopy
  • Pichia pastoris
  • Relaxation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Dikiy, I., Clark, L. D., Gardner, K. H., & Rosenbaum, D. M. (2019). Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics. In A. J. Wand (Ed.), Methods in Enzymology (pp. 37-65). (Methods in Enzymology; Vol. 614). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.08.030