Single Molecule Tracking of Annexin V in Cushioned DMPC Assemblies

Abstract

Using single molecule imaging, this study describes the phase behavior and mobility of individual transmembrane (TM) proteins and compares those results with the bulk phase behavior of the biomimetic membrane in which they have been incorporated. To accomplish this a TM protein, Annexin V, was incorporated into a cushioned planar supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) / L-α-lysophosphatidyl-serine (Brain-PS) biomimetic assembly and its mobility between 30 and 16 C was measured. Fluorescence microscopy and Fluorescence Recovery After Photobleaching (FRAP) were used to verify the structural integrity and the phase behavior (median melting temperature, TC = 22 C) of the lipid assembly. The spatial confinement of individual Annexin V molecules was measured in three distinct phase regions: (1) a homogenous liquid crystalline phase region (Lα) in which Annexin V was unconfined (≥ 25 C), (2) a two-phase region (Lα + gel-phase-Pβ′) in which Annexin V displayed intermediate confinement (24 - 20 C), and (3) a gel-phase region (Pβ′) with included nanoscopic domains that are enriched with PS and surround a single Annexin V TM protein (19 - 16 C); the mobility of Annexin V in these domains is highly confined. At early time lags, Annexin V moves with apparent Brownian-like behavior at all temperatures but the diffusion coefficients have very different magnitudes and temperature dependence. A possible mechanism for nanoscopic domain formation will be discussed.