Assembly of Membrane-Bound Protein Complexes: Detection and Analysis by Single Molecule Diffusion

Abstract

Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca2+-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves and quantitates the rapidly diffusing monomer and slowly diffusing heterodimer subpopulations. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing average diffusion coefficient of the fluorescent PH-CaM population, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ∼102-fold greater in the membrane system than in solution, apparently due both to faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.

Lateral diffusion of peripheral membrane proteins on supported lipid bilayers is controlled by the additive frictional drags of (1) bound lipids and (2) protein domains penetrating into the bilayer hydrocarbon core

“…As in previous studies, single molecule diffusion tracks were recorded with a 20 msec frame rate, and populations of hundreds of tracks were analyzed (11–13). A significant advantage of the single molecule analysis is the ability to resolve diffusional heterogeneity and to filter out minor population components exhibiting anomalous diffusion, thereby more accurately defining the diffusion constant of the major component.…”

“…Previous exploratory studies have indicated that 2-dimensional diffusion rates of peripheral membrane proteins on planar supported bilayers provide a new window into (i) protein-lipid, (ii) protein-bilayer, and (iii) protein-protein interactions (10–13). Each of these interactions can trigger a significant, detectable change in diffusion speed on the supported bilayer surface that provides key insights into a molecular interaction or event.…”

“…Moreover, the unique boundary conditions and interactions of one leaflet with the glass support may expand the dynamic range of lipid and protein diffusion constants in supported bilayers (15,16). Taken together, the characteristic features of supported bilayers make them especially well suited for single molecule mechanistic studies that detect and analyze molecular events based on changes of diffusion speed (11–13). Such diffusional changes can be used to monitor, for example, the events that occur as membrane-recruited signaling proteins become activated by the stepwise binding of activator lipids, regulatory proteins, and substrates.…”

Cadherin Diffusion in Supported Lipid Bilayers Exhibits Calcium-Dependent Dynamic Heterogeneity

“…After incubating for 2 h at room temperature, liposomes fused on the substrate, and spontaneously formed a supported lipid bilayer (30,31). The bilayer was rinsed thoroughly with water to remove excess liposomes and protein, and then exchanged into HEPES/CaCl 2 buffer at the desired Ca 2þ concentration (32,33). For control experiments, pure DOPC lipid bilayers and DOPC lipid bilayers containing a low concentration of fluorescent DOPE-LR as probes were prepared using the procedure described above.…”

Interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells

“…The lipid bilayer plays a central role in this network by serving as a shared platform for two-dimensional diffusion, thereby enabling the membrane-bound signaling proteins to encounter and bind their membrane-associated, lipid and/or protein effectors and substrates (Ziemba et al, 2013; Ziemba and Falke, 2013; Ziemba et al, 2012; Knight et al, 2010; Knight and Falke, 2009). The resulting, membrane-localized signaling circuit controls actin polymerization, membrane remodeling, and other dynamic processes essential for growth of the leading edge up attractant gradients.…”