Beyond Saffman-Delbruck approximation: A new regime for 2D diffusion of α-hemolysin complexes in supported lipid bilayer

Abstract

Cell mechanisms are actively modulated by membrane dynamics. We studied the dynamics of a first-stage biomimetic system by Fluorescence Recovery After Patterned Photobleaching. Using this simple biomimetic system, constituted by α -hemolysin from Staphylococcus aureus inserted as single heptameric pore or complexes of pores in a glass-supported DMPC bilayer, we observed true diffusion behavior, with no immobile fraction. We find two situations: i) when incubation is shorter than 15 hours, the protein inserts as a heptameric pore and diffuses roughly three times more slowly than its host lipid bilayer; ii) incubation longer than 15 hours leads to the formation of larger complexes which diffuse more slowly. Our results indicate that, while the Saffman-Delbruck model adequately describes the diffusion coefficient D for small radii, D of the objects decreases as 1/R(2) for the size range explored in this study. Additionally, in the presence of inserted proteins, the gel-to-fluid transition of the supported bilayer as well as a temperature shift in the gel-to-fluid transition are observed.

Transmembrane Protein Diffusion in Gel-Supported Dual-Leaflet Membranes

“…Interpreting experiments is challenging, and has been controversial, because there are many factors that influence measured tracer mobilities (5,17,18,(23)(24)(25). At the highest level, these factors include the measurement method (fluorescence recovery after photobleaching, nuclear magnetic resonance, and/or fluorescence correlation spectroscopy); the tracer and its fluorescent tag; the membrane and its composition; the support and its membrane synthesis; and the buffer.…”

“…Such a scaling is predicted by dissipative particle dynamics (coarse-grained) computations (26), but only at short to intermediate timescales, and for inclusions, such as lipid rafts, in solvent-supported membranes. Note that the scaling hinges on the inclusions having internal lateral degrees of freedom, so is unlikely to apply to the experiments of Harb et al (25), which were undertaken with mono-, di-, and trimeric assemblies of heptomeric pores using fluorescence recovery after photobleaching, thus probing diffusion on long timescales.…”

Electric migration of α‐hemolysin in supported n‐bilayers: A model for transmembrane protein microelectrophoresis

“…After insertion into the bilayer, this should lead, on average, to one monomer per heptamer. Analysis of the fluorescence level of spots using Image J confirms a narrow labeling distribution (for details see ).…”

“…For the purpose of protein insertion, SLBs were incubated with an excess of α‐Hl at a final concentration of 0.1 μM in 10 mM phosphate buffer pH 7.2 and 100 mM NaCl for different lengths of time (from 18 up to 48 h) . Starting with water‐soluble monomer, which forms the heptameric pore once inserted, greatly simplifies this step; here, it enabled all proteins to be unidirectionally incorporated in the lipid bilayer.…”

“…We showed in a previous work that the interaction of α‐hemolysin (α‐Hl) with glass, after stereo‐specific insertion, is sufficiently weak to avoid its immobilization. Fluorescence recovery after patterned photobleaching (FRAPP) experiments revealed that the inserted heptameric pore had a diffusion coefficient compatible with an embedded object of a radius close to 5 nm, while incubation in the same conditions over an SLB on mica, probably due to a higher interaction with the support (different zeta potentials, different number of interaction sites due to differing roughness between glass and mica), led to no diffusion of the heptamers.…”