Single-molecule tweezers decoding hidden dimerization patterns of membrane proteins within lipid bilayers

Dimerization of transmembrane (TM) proteins is an essential biological process within cellular membranes, playing a key role in diverse pathophysiological pathways and serving as a promising therapeutic target. Although often simplified as a two-state transition from freely diffusing monomers to fully formed dimers, the dimerization process after monomer diffusion—the post-diffusion dimerization—is likely more complex due to intricate inter-residue interactions. Here, we introduce a single-molecule tweezer platform to map detailed profiles of the post-diffusion transitions in TM protein dimerization. This approach captures reversible dimerization events of a single TM dimer, revealing hidden intermediate states that emerge following the quiescent phase of monomer diffusion. Profiling the post-diffusion intermediates, kinetics, and energy landscapes—integrated with molecular dynamics simulations—uncovers the dimerization pathway, the effects of residue interactions and lipid bilayers, and the kinetic and energetic contributions of distinct dimerization domains. Furthermore, this platform characterizes selective and localized modulations via peptide binding, underscoring its potential to elucidate the mechanisms of action of TM dimer-targeting drugs at single-molecule resolution.