To investigate how human dystrophin associates with biomimetic phospholipid bicelles, we applied switchSENSE®, a fluorescence-based biosensing technology that employs dynamic DNA nanolevers in a microfluidic environment with minimal material requirements. We show that switchSENSE® enables real-time kinetic analysis of weak and transient interactions, offering a clear advantage over classical biophysical methods that often fail to capture low-affinity protein-lipid binding. Focusing on two functionally relevant central-domain dystrophin fragments (R1-3 and R11-15), we quantified binding to zwitterionic and anionic bicelles using two complementary assay orientations: proteins immobilized with bicelles as analytes, and the reverse. Both yielded consistent micromolar affinities, in line with prior microscale thermophoresis results, but uniquely provided kinetic rate constants. This represents the first kinetic characterization of dystrophin-bicelle interactions, revealing that weak affinity is largely driven by rapid dissociation. Notably, R11-15 exhibited faster association and slower dissociation than R1-3, indicating a more stable and sustained lipid interaction. Beyond advancing mechanistic insight into dystrophin’s reversible membrane association – a process thought to stabilize sarcolemmal integrity during muscle contraction and elongation -, our study highlights switchSENSE® as a versatile platform for quantifying weak protein-lipid interactions. By enabling kinetic resolution of interactions at the edge of conventional detection limits, switchSENSE® provides a powerful tool for dissecting the dynamic interplay between structural proteins and lipid assemblies.
Combet et al. (Mon,) studied this question.