TY - JOUR

T1 - Solution NMR spectroscopy of GPCRs

T2 - Residue-specific labeling strategies with a focus on 13C-methyl methionine labeling of the atypical chemokine receptor ACKR3

AU - Kleist, Andrew B

AU - Peterson, Francis

AU - Tyler, Robert C

AU - Gustavsson, Martin

AU - Handel, Tracy M

AU - Volkman, Brian F

N1 - © 2019 Elsevier Inc. All rights reserved.

PY - 2019

Y1 - 2019

N2 - The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques. Despite these advantages, NMR-based studies of GPCRs are few relative to the number of published structures, due in part to the challenges and limitations of NMR for the characterization of large membrane proteins. Several studies have circumvented these challenges using a variety of isotopic labeling strategies, including side chain derivatization and metabolic incorporation of NMR-active nuclei. In this chapter, we provide an overview of different isotopic labeling strategies and describe an in-depth protocol for the expression, purification, and NMR studies of the chemokine GPCR atypical chemokine receptor 3 (ACKR3) via 13CH3-methionine incorporation. The goal of this chapter is to provide a resource to the GPCR community for those interested in pursuing NMR studies of GPCRs.

AB - The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques. Despite these advantages, NMR-based studies of GPCRs are few relative to the number of published structures, due in part to the challenges and limitations of NMR for the characterization of large membrane proteins. Several studies have circumvented these challenges using a variety of isotopic labeling strategies, including side chain derivatization and metabolic incorporation of NMR-active nuclei. In this chapter, we provide an overview of different isotopic labeling strategies and describe an in-depth protocol for the expression, purification, and NMR studies of the chemokine GPCR atypical chemokine receptor 3 (ACKR3) via 13CH3-methionine incorporation. The goal of this chapter is to provide a resource to the GPCR community for those interested in pursuing NMR studies of GPCRs.

KW - Animals

KW - Carbon Isotopes/chemistry

KW - Cell Line

KW - Humans

KW - Magnetic Resonance Spectroscopy/methods

KW - Receptors, CXCR/metabolism

KW - Receptors, G-Protein-Coupled/metabolism

KW - Solutions

KW - Staining and Labeling

KW - Vitamin U/metabolism

U2 - 10.1016/bs.mcb.2018.09.004

DO - 10.1016/bs.mcb.2018.09.004

M3 - Journal article

C2 - 30616824

VL - 149

SP - 259

EP - 288

JO - Methods in Cell Biology

JF - Methods in Cell Biology

SN - 0091-679X

ER -