Enhanced site-specific fluorescent labeling of membrane proteins using native nanodiscs

Fluorescent labeling of membrane proteins is essential for exploring their functions, signaling pathways, interaction partners, and structural dynamics. Organic fluorophores are commonly used for this purpose due to their favorable photophysical properties and photostability. However, a persistent challenge is the inaccessibility of surface-exposed cysteine residues required for site-specific labeling, as these residues often become sequestered within detergent micelles during protein extraction. To address this limitation, we developed an approach based on polymer-encapsulated nanodiscs that preserves the protein’s native-like lipid-bilayer environment while ensuring accessibility of surface-exposed cysteine residues. In this method, His-tagged proteins embedded in native nanodiscs are retained on a nickel affinity column, allowing for simultaneous purification and labeling by adding fluorescent dyes. This versatile technique was demonstrated with two challenging-to-label membrane proteins, the potassium channel KvAP and the urea channel HpUreI, for which detergent-based labeling had failed. It opens new possibilities for studying a wide range of fluorescently labeled membrane proteins in near-native states, advancing applications in biophysics, structural biology, and drug discovery.