Author:
Nakagawa Fuhito,Kikkawa Marin,Chen Sisi,Miyashita Yasuomi,Hamaguchi-Suzuki Norie,Shibuya Minami,Yamashita Soichi,Nagase Lisa,Yasuda Satoshi,Shiroishi Mitsunori,Senda Toshiya,Ito Keisuke,Murata Takeshi,Ogasawara Satoshi
Abstract
AbstractNanodisc technology has dramatically advanced the analysis of molecular interactions for membrane proteins. A nanodisc is designed as a vehicle for membrane proteins that provide a native-like phospholipid environment and better thermostability in a detergent-free buffer. This enables the determination of the thermodynamic and kinetic parameters of small molecule binding by surface plasmon resonance. In this study, we generated a nanodisc specific anti-MSP (membrane scaffold protein) monoclonal antibody biND5 for molecular interaction analysis of nanodiscs. The antibody, biND5 bound to various types of nanodiscs with sub-nanomolar to nanomolar affinity. Epitope mapping analysis revealed specific recognition of 8 amino acid residues in the exposed helix-4 structure of MSP. Further, we performed kinetics binding analysis between adenosine A2a receptor reconstituted nanodiscs and small molecule antagonist ZM241385 using biND5 immobilized sensor chips. These results show that biND5 facilitates the molecular interaction kinetics analysis of membrane proteins substituted in nanodiscs.
Funder
Japan Agency for Medical Research and Development
Core Research for Evolutional Science and Technology
Japan Society for the Promotion of Science
Chiba University
Publisher
Springer Science and Business Media LLC
Cited by
1 articles.
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