Approaching protein-protein interactions in membranes using single-particle tracking and packing coefficient analysis

Abstract

Molecules diffuse randomly in the plane of the plasma membrane due to membrane fluidity. However, interactions with other molecules may introduce noticeable changes in diffusion behavior such as a slowdown or immobilization by formation of complexes. As a consequence, the analysis of transitions between diffusive behaviors can provide effective rate constants of molecular interactions. Tracking the positions of labeled single molecules, i.e., using single-particle tracking (SPT), is particularly pertinent for this kind of studies. We provide here a step-by-step protocol for SPT experiments and analyses needed to estimate the effective rate constants of molecular interactions. Classical SPT data analyses using the mean square displacement provide the average diffusive behavior, thus precluding the analysis of transitions. We propose an alternative approach to overcome this problem, namely the packing coefficient (Pc) analysis. We illustrate the application of this method to the interactions of neurotransmitter receptors with their scaffolding proteins.