Characterization of membrane protein-nanobody complexation via flow-induced dispersion analysis (FIDA)

Nanobody binders may be raised against membrane protein targets for a myriad of applications including the modulation of bioactivity and to aid in structural elucidation. Flow-Induced Dispersion Analysis (FIDA) is a burgeoning analytical technique which has been used to quantify protein-protein binding interactions. FIDA offers a straightforward route to characterizing membrane protein-nanobody complexation in terms of affinity and particle size. A protocol for the rapid characterisation of nanobodies that bind membrane proteins with high affinity using FIDA would expedite the development of novel nanobody-based biomedicines and facilitate nanobody-assisted membrane protein structural biology. In this study, FIDA was used to characterize the complexation of a nanobody and a model membrane integral protein transporter. To this end, the nanobody was fluorescently labelled using fluorescein isothiocyanate. A simple two-step protocol for producing labelled nanobody stocks devoid of interfering free dye is presented. FIDA experiments entailing sequential addition of the micellarised transporter protein to the labelled nanobody in detergent-containing buffer revealed single digit nanomolar binding consistent with binding constants obtained previously by surface plasmon resonance analysis.