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

6 Citations (Scopus)

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

Fingerprint

Pichia
G-Protein-Coupled Receptors
Labeling
Membrane Proteins
Nuclear magnetic resonance
Ligands
Isoleucine
Yeast
Labels
Yeasts
Lipids
Pharmaceutical Preparations
Proteins

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

Cite this

On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies. / Clark, Lindsay; Dikiy, Igor; Rosenbaum, Daniel M.; Gardner, Kevin H.

In: Journal of Biomolecular NMR, Vol. 71, No. 4, 01.08.2018, p. 203-211.

Research output: Contribution to journalArticle

@article{a339ff8bf7d14bc2ba145f5ee0334a2f,
title = "On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies",
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.",
keywords = "Deuteration, GPCR, Integral membrane proteins, Pichia pastoris, Solution NMR, Stable isotope labeling",
author = "Lindsay Clark and Igor Dikiy and Rosenbaum, {Daniel M.} and Gardner, {Kevin H.}",
year = "2018",
month = "8",
day = "1",
doi = "10.1007/s10858-018-0204-3",
language = "English (US)",
volume = "71",
pages = "203--211",
journal = "Journal of Biomolecular NMR",
issn = "0925-2738",
publisher = "Springer Netherlands",
number = "4",

}

TY - JOUR

T1 - On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies

AU - Clark, Lindsay

AU - Dikiy, Igor

AU - Rosenbaum, Daniel M.

AU - Gardner, Kevin H.

PY - 2018/8/1

Y1 - 2018/8/1

N2 - 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.

AB - 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.

KW - Deuteration

KW - GPCR

KW - Integral membrane proteins

KW - Pichia pastoris

KW - Solution NMR

KW - Stable isotope labeling

UR - http://www.scopus.com/inward/record.url?scp=85050583224&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85050583224&partnerID=8YFLogxK

U2 - 10.1007/s10858-018-0204-3

DO - 10.1007/s10858-018-0204-3

M3 - Article

C2 - 30121871

AN - SCOPUS:85050583224

VL - 71

SP - 203

EP - 211

JO - Journal of Biomolecular NMR

JF - Journal of Biomolecular NMR

SN - 0925-2738

IS - 4

ER -