Kinetics of Membrane Bending by Protein Crowding

Abstract

Dynamic shape changes in phospholipid membranes are considered critical to a number of important cellular processes. Such structural changes are thought to be driven by a number of possible mechanisms. Recently, the effects of protein crowding on shaping and remodeling membrane surfaces has attracted a lot of interest due to the ubiquity of crowded interfaces in biological systems. While a number of recent studies have confirmed the ability of proteins to drive curvature solely via a colligative, crowding-based mechanism- a number of key fundamental mechanistic details remain unsolved. Specifically, very little is known about the kinetics of this process. What are the timescales of membrane deformation process? How kinetically separated are protein binding and the membrane bending events? Is membrane deformation preceded by a previously unresolved intermediate? To address these questions, we provide a comprehensive study on the kinetics of these events through stopped-flow fluorescence using FRET as the spectroscopic handle. A fluorophore-quencher pair was used to study the protein binding kinetics to the bilayer, while a donor-acceptor pair was used as a reporter for membrane area expansion. We report a complex multi-phasic kinetic behavior for both the protein association and membrane expansion process. The association rates follow a surprising negative trend with respect to increasing protein concentration presumably due to saturation of membrane binding sites owing to protein crowding. We find that the membrane deformation happens at significantly slower timescales relative to the protein binding events. The complexity of the fits suggest that the membrane bending process might be preceded by an unresolved intermediate. Taken together with a predictive theoretical model that is currently underway, we believe that our observations will advance our knowledge about membrane bending driven by protein crowding.