On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies

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

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.

Original languageEnglish (US)
Pages (from-to)203-211
Number of pages9
JournalJournal of biomolecular NMR
Volume71
Issue number4
DOIs
StatePublished - Aug 1 2018

Keywords

  • Deuteration
  • GPCR
  • Integral membrane proteins
  • Pichia pastoris
  • Solution NMR
  • Stable isotope labeling

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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