Journal
PEERJ
Volume 10, Issue -, Pages -Publisher
PEERJ INC
DOI: 10.7717/peerj.13381
Keywords
GPCR; Dissociation constant; Detergents; Styrene maleic acid copolymer nanodiscs; Smoothened receptors; NMR
Categories
Funding
- Foundation of Anhui Technologies Major Program [201903a07020026]
- Foundation of President of the Chinese Academy of Sciences [YZJJZX202004]
- National Natural Science Foundation of China [32171226]
- High Magnetic Field Laboratory of Anhui Province
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In this study, we used styrene maleic acid (SMA) copolymer to extract and purify the SMO transmembrane domain, encapsulated by SMA-nanodiscs, maintaining the physiological activity of SMO protein. The obtained SMA-nanodiscs showed high homogeneity. This work paves the way for investigating the structure, function, and drug development of SMO proteins in a native-like lipid environment.
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 (K-d) 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.
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