Suspended lipid bilayer for optical and electrical measurements of single ion channel proteins.

Making and holding an artificial lipid bilayer horizontally in an aqueous solution within the microscopic working distance of ~100 µm are essential for simultaneous single molecule imaging and single ion-channel electrical current recording. However, preparation of such a lipid bilayer without a solid support is technically challenging. In a typical supported lipid bilayer, the asymmetric local environments and the strong perturbation of the underneath solid or dense surface can diverge the normal behavior of membrane proteins and lipids. On the other hand, the suspended lipid bilayers can provide a native local environment for the membrane proteins and lipids by having fluids on both sides. In this technical report, we present a simple and novel methodology for making a suspended lipid bilayer that can be used for recording the single-molecule diffusion and single ion-channel electrical measurements of ion-channel proteins. Our approach has a higher validity for studying the molecular diffusions and conformational fluctuations of membrane proteins without having perturbations from supporting layers. We demonstrate the feasibility of such an approach on simultaneous single-molecule fluorescence imaging and electric current measurements of ion channel proteins.

Combined topographic, spectroscopic, and model analyses of inhomogeneous energetic coupling of linear light harvesting complex II aggregates in native photosynthetic membranes.

Light harvesting by LH1 and LH2 antenna proteins in the photosynthetic membranes of purple bacteria has been extensively studied in recent years for the fundamental understanding of the energy transfer dynamics and mechanism. Here we report the inhomogeneous structural organization of the LH2 complexes in photosynthetic membranes, giving evidence for the existence of energetically coupled linear LH2 aggregates in the native photosynthetic membranes of purple bacteria. Focusing on systematic model analyses, we combined AFM imaging and spectroscopic analysis with energetic coupling model analysis to characterize the inhomogeneous linear aggregation of LH2. Our AFM imaging results reveal that the LH2 complexes form linear aggregates with the monomer number varying from one to eight and each monomer tilted along the aggregated structure in photosynthetic membranes. The spectroscopic results support the attribution of aggregated LH2 complexes in the photosynthetic membranes, and the model calculation values for the absorption, emission and lifetime are consistent with the experimentally determined spectroscopic values, further proving a molecular-level understanding of the energetic coupling and energy transfer among the LH2 complexes in the photosynthetic membranes.

Anticorrelated position fluctuation of lipids in forming membrane water pores: molecular dynamics simulations study with dengue virus capsid protein.

The traffic of molecules into or out of cells is regulated by many membrane-associated mechanisms. Membrane pores are considered as one of the major passage mechanisms, although molecular-level understanding of pore formation is still vague. The opening of a membrane pore depends on many factors, including the influence of some proteins. The ability of the cell-penetrating peptides and supercharged proteins to form membrane pores has been reported. We studied pore formation through dipalmitoylphosphatidylcholine (DPPC) lipid bilayers by supercharged dengue virus capsid (C) protein. Atomistic molecular dynamics simulations confirmed the formation of membrane pores by a combined effect of the C protein and the membrane electric field. Analyses of simulated trajectories showed highly correlated vertical position fluctuations between the Cα atom of the membrane-anchored arginine residues and the phosphorus atoms of the surrounding DPPC lipids. Certain regions of the bilayer were negatively correlated while the others were positively correlated with respect to the fluctuations of the Cα atom of the anchored arginine residues. When positively correlated lipids in one leaflet vertically aligned with the negatively correlated lipids in the other leaflet, a local anticorrelated region was generated by weakening the bilayer. The membrane pore was always formed close to this anticorrelated region. Once formed, the C protein followed the hydrated pathway provided by the water-filled pores to cross the membrane.Communicated by Ramaswamy H. Sarma.

Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane.

We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ∼15 Šdiameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a lipid bilayer.