Effective application of bicelles for conformational analysis of G protein-coupled receptors by hydrogen/deuterium exchange mass spectrometry.
G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.
School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, South Korea.
Nguyen Minh
NM
Duc
Department of Molecular and Cellular Physiology, Stanford University, Medical School, 297 Campus Drive, Beckman Center, Stanford, California, USA.
Yang
Y
Du
Department of Molecular and Cellular Physiology, Stanford University, Medical School, 297 Campus Drive, Beckman Center, Stanford, California, USA.
Cheng
C
Zhang
School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, South Korea.
Su Youn
SY
Lee
Department of Molecular and Cellular Physiology, Stanford University, Medical School, 297 Campus Drive, Beckman Center, Stanford, California, USA.
Thor S
TS
Thorsen
Department of Molecular and Cellular Physiology, Stanford University, Medical School, 297 Campus Drive, Beckman Center, Stanford, California, USA.
Brian K
BK
Kobilka
School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, South Korea.
Ka Young
KY
Chung
D000595:Amino Acid Sequence; D003902:Detergents / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D005240:Feasibility Studies; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
C553443:ADRB2 protein, human; D003902:Detergents; D008051:Lipid Bilayers; D008823:Micelles; C485764:OPRM1 protein, human; D044463:Receptor, PAR-1; D018343:Receptors, Adrenergic, beta-2; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; C411362:dodecyl maltopyranoside; D008320:Maltose