![]() ![]() ![]() Since nanodiscs are without detergents and both the inside and outside regions of the membrane protein are exposed to aqueous solution, it is possible to analyze the interactions of ligands in solution, such as membrane-associating proteins, with the surface of the lipid bilayer. Nanodiscs are an attractive alternative for solubilizing membrane proteins and have the added advantages of maintaining the lipid bilayer environment and improving thermal stability.Ī nanodisc is one format of membrane protein, composed of discoidal lipid bilayers of phospholipids surrounded by amphipathic molecules, such as a membrane scaffold proteins (MSPs) (Fig. Thus, study protocols that require detergents may not provide a sufficiently stable environment for intermolecular interaction analysis, especially that of small molecule ligands 4, 5, 6, 7. However, purification using detergents creates a highly dynamic environment wherein there is constant exchange of the detergent micelles surrounding the membrane proteins with those in the buffer. Membrane proteins are extracted from the cell membrane using detergents that are also used in subsequent purification and analysis processes. Surface plasmon resonance (SPR) is a well-established method for label-free evaluation of protein–ligand interactions in real-time with kinetics parameters such as association rate constants (k on) and dissociation rate constants (k off) 2, 3. Since most of these physiological processes involve the interaction of target proteins with small molecule ligands, the analysis of interactions between ligand and membrane proteins and screening of ligand libraries is crucial in drug discovery research. Membrane proteins play key roles in various physiological processes, such as signal transduction, substance transport, enzyme catalysis, synaptic transmission regulation, and immune response, and are therefore, targets of more than 50% of therapeutic drugs 1. These results show that biND5 facilitates the molecular interaction kinetics analysis of membrane proteins substituted in nanodiscs. Further, we performed kinetics binding analysis between adenosine A 2a receptor reconstituted nanodiscs and small molecule antagonist ZM241385 using biND5 immobilized sensor chips. Epitope mapping analysis revealed specific recognition of 8 amino acid residues in the exposed helix-4 structure of MSP. The antibody, biND5 bound to various types of nanodiscs with sub-nanomolar to nanomolar affinity. In this study, we generated a nanodisc specific anti-MSP (membrane scaffold protein) monoclonal antibody biND5 for molecular interaction analysis of nanodiscs. ![]() This enables the determination of the thermodynamic and kinetic parameters of small molecule binding by surface plasmon resonance. 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. Nanodisc technology has dramatically advanced the analysis of molecular interactions for membrane proteins. ![]()
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