Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer
Smoothened (SMO) protein is a member of the G protein-coupled receptor (GPCR) family that is involved in the Hedgehog (Hh) signaling pathway. It is a putative target for treating various cancers, including medulloblastoma and basal cell carcinoma (BCC). Characterizing membrane proteins such as SMO i...
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PeerJ Inc.
2022-05-01
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| Online Access: | https://peerj.com/articles/13381.pdf |
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| author | Lina Zhu Hongxin Zhao Yizhuo Wang Chuandi Yu Juanjuan Liu Ling Li Zehua Li Jin Zhang Han Dai Junfeng Wang Lei Zhu |
| author_facet | Lina Zhu Hongxin Zhao Yizhuo Wang Chuandi Yu Juanjuan Liu Ling Li Zehua Li Jin Zhang Han Dai Junfeng Wang Lei Zhu |
| author_sort | Lina Zhu |
| collection | DOAJ |
| description | Smoothened (SMO) protein is a member of the G protein-coupled receptor (GPCR) family that is involved in the Hedgehog (Hh) signaling pathway. It is a putative target for treating various cancers, including medulloblastoma and basal cell carcinoma (BCC). Characterizing membrane proteins such as SMO in their native state is highly beneficial for the development of effective pharmaceutical drugs, as their structures and functions are retained to the highest extent in this state. Therefore, although SMO protein is conventionally solubilized in detergent micelles, incorporating the protein in a lipid-based membrane mimic is still required. In this study, we used styrene maleic acid (SMA) copolymer that directly extracted membrane protein and surrounding lipids as well as formed the so-called polymer nanodiscs, to solubilize and purify the SMO transmembrane domain encapsulated by SMA-nanodiscs. The obtained SMA-nanodiscs showed high homogeneity and maintained the physiological activity of SMO protein, thereby enabling the measurement of the dissociation constant (Kd) for SMO ligands SMO-ligands Shh Signaling Antagonist V (SANT-1) and Smoothened Agonist (SAG) using ligand-based solution nuclear magnetic resonance spectroscopy. This work paves the way for investigating the structure, function, and drug development of SMO proteins in a native-like lipid environment. |
| first_indexed | 2024-03-09T08:19:25Z |
| format | Article |
| id | doaj.art-4793618d30f14628818d260319f7bc83 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T08:19:25Z |
| publishDate | 2022-05-01 |
| publisher | PeerJ Inc. |
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| series | PeerJ |
| spelling | doaj.art-4793618d30f14628818d260319f7bc832023-12-02T21:53:31ZengPeerJ Inc.PeerJ2167-83592022-05-0110e1338110.7717/peerj.13381Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymerLina Zhu0Hongxin Zhao1Yizhuo Wang2Chuandi Yu3Juanjuan Liu4Ling Li5Zehua Li6Jin Zhang7Han Dai8Junfeng Wang9Lei Zhu10Institute of Physical Science and Information Technology, Anhui University, Hefei, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaSchool of Basic Medical Sciences, Nanchang University, Nanchang, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaInstitute of Physical Science and Information Technology, Anhui University, Hefei, ChinaHigh Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaSmoothened (SMO) protein is a member of the G protein-coupled receptor (GPCR) family that is involved in the Hedgehog (Hh) signaling pathway. It is a putative target for treating various cancers, including medulloblastoma and basal cell carcinoma (BCC). Characterizing membrane proteins such as SMO in their native state is highly beneficial for the development of effective pharmaceutical drugs, as their structures and functions are retained to the highest extent in this state. Therefore, although SMO protein is conventionally solubilized in detergent micelles, incorporating the protein in a lipid-based membrane mimic is still required. In this study, we used styrene maleic acid (SMA) copolymer that directly extracted membrane protein and surrounding lipids as well as formed the so-called polymer nanodiscs, to solubilize and purify the SMO transmembrane domain encapsulated by SMA-nanodiscs. The obtained SMA-nanodiscs showed high homogeneity and maintained the physiological activity of SMO protein, thereby enabling the measurement of the dissociation constant (Kd) for SMO ligands SMO-ligands Shh Signaling Antagonist V (SANT-1) and Smoothened Agonist (SAG) using ligand-based solution nuclear magnetic resonance spectroscopy. This work paves the way for investigating the structure, function, and drug development of SMO proteins in a native-like lipid environment.https://peerj.com/articles/13381.pdfGPCRDissociation constantDetergentsStyrene maleic acid copolymer nanodiscsSmoothened receptorsNMR |
| spellingShingle | Lina Zhu Hongxin Zhao Yizhuo Wang Chuandi Yu Juanjuan Liu Ling Li Zehua Li Jin Zhang Han Dai Junfeng Wang Lei Zhu Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer PeerJ GPCR Dissociation constant Detergents Styrene maleic acid copolymer nanodiscs Smoothened receptors NMR |
| title | Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer |
| title_full | Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer |
| title_fullStr | Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer |
| title_full_unstemmed | Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer |
| title_short | Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer |
| title_sort | solubilization purification and ligand binding characterization of g protein coupled receptor smo in native membrane bilayer using styrene maleic acid copolymer |
| topic | GPCR Dissociation constant Detergents Styrene maleic acid copolymer nanodiscs Smoothened receptors NMR |
| url | https://peerj.com/articles/13381.pdf |
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