Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Protein kinase C penetration into lipid bilayers

Physical characteristics of the association and subsequent penetration of protein kinase C into defined lipid bilayers were analyzed using four different fluorescence probes. The enzyme demonstrated strong hydrophobic and electrostatic interactions with the bilayer as suggested by its ability to increase permeability of carboxyfluorescein-filled unilamellar vesicles. The intensity of interaction was dependent on the concentration of phosphatidylserine. The hydrophilic quencher, N-methylpicolinium perchlorate, was used to show that the tryptophan residues affected by ligand-induced conformational changes were in a hydrophobic region(s) of the enzyme. Using quenching of intrinsic tryptophan fluorescence, the enzyme was shown to penetrate the lipid bilayer to the C-16 position of labeled fatty acid probes. The association and subsequent penetration of the enzyme into the lipid bilayer was independent of divalent cations in these systems and had no significant effect on activator-independent substrate phosphorylation.     

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.     

Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.     

Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers

One of the biggest challenges in pharmaceutical research is obtaining integral membrane proteins in a functional, solubilized, and monodisperse state that provides a native-like environment that maintains the spectrum of in vivo activities. Many of these integral membrane proteins are receptors, enzymes, or other macromolecular assemblies that are important drug targets. An example is the general class of proteins composed of seven-transmembrane segments (7-TM) as exemplified by the G-protein-coupled receptors. In this article, we describe a simple system for self-assembling bacteriorhodopsin, as a model protein containing 7-TM helices, with phospholipids to form a nanometer-scale soluble bilayer structure encircled by a 200 amino acid scaffold protein. The result is the single molecule incorporation of an integral membrane protein target into a soluble and monodisperse structure that allows the structural and functional tools of solution biochemistry to be applied.     

Reconstitution of membrane proteins: sequential incorporation of integral membrane proteins into preformed lipid bilayers

Several integral membrane proteins can be inserted sequentially into preformed unilamellar vesicles (ULV's) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol in a gel phase. Thus, proteoliposomes of DMPC, cholesterol, and bacteriorhodopsin from Halobacterium halobium rapidly incorporate UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase from beef heart mitochondria, and additional bacteriorhodopsin, added sequentially. This process of spontaneous incorporation can be regulated to produce complex artificial membranes that contain phospholipids and proteins at ratios (mol/mol) equivalent to what is found in biological membranes. The ability of the lipid-protein bilayers to incorporate additional integral membrane proteins is not affected by annealing of the proteoliposomes at 37 degrees C nor by the order of addition of the proteins. Bacteriorhodopsin-containing vesicles formed by the sequential addition of integral membrane proteins demonstrate light-driven proton pumping. Therefore, they have retained a vesicular structure. Vesicles containing one or two different proteins will fuse with each other at 21 degrees C or with ULV's devoid of proteins. Incorporation of bacteriorhodopsin or UDPglucuronosyltransferase into proteoliposomes containing DMPC, with or without cholesterol as impurity, also occurs above the phase transition for DMPC. The presence of a protein in a liquid-crystalline bilayer provides the necessary condition for promoting the spontaneous incorporation of other membrane proteins into preformed bilayers.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.     

Ion channels from synaptic vesicle membrane fragments reconstituted into lipid bilayers.

Cholinergic synaptic vesicles were isolated from the electric organ of Torpedo californica. Vesicle membrane proteins were reconstituted into planar lipid bilayers by the nystatin/ergosterol fusion technique. After fusion, a variety of ion channels were observed. Here we identify four channels and describe two of them in detail. The two channels share a conductance of 13 pS. The first is anion selective and strongly voltage dependent, with a 50% open probability at membrane potentials of -15 mV. The second channel is slightly cation selective and voltage independent. It has a high open probability and a subconductance state. A third channel has a conductance of 4-7 pS, similar to the subconductance state of the second channel. This channel is fairly nonselective and has gating kinetics different from those of the cation channel. Finally, an approximately 10-pS, slightly cation selective channel was also observed. The data indicate that there are one or two copies of each of the above channels in every synaptic vesicle, for a total of six channels per vesicle. These observations confirm the existence of ion channels in synaptic vesicle membranes. It is hypothesized that these channels are involved in vesicle recycling and filling.     

Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers

One of the biggest challenges in the field of proteomics is obtaining functional membrane proteins solubilized and dispersed into a physiologically relevant environment that maintains the spectrum of in vivo activities. Here we describe a system composed of nanoscale self-assembled particles, termed Nanodiscs, which contain a single phospholipid bilayer stabilized by an encircling membrane scaffold protein (MSP). Using microsomal membranes of baculovirus-infected Spodoptera frugiperda (Sf9) insect cells overexpressing an N-terminally anchored cytochrome P450 monoxygenase (P450), we demonstrate that target membrane proteins can be directly solubilized and incorporated into distinct populations of Nanodiscs, which can be separated by size chromatography. We show that formation of these Nanodiscs from insect cell membranes allows for the compartmentalization into soluble nanostructures that provide a natural membrane bilayer that avoids the aggregation of membrane proteins often encountered in other reconstitution procedures. Lipid composition analysis and substrate binding analysis of size-fractionated Nanodiscs arrayed in microtiter plates further demonstrates that the Nanodisc system effectively disperses the overexpressed membrane protein into monodispersed bilayers containing biochemically defined lipid components and the target protein in its native from suitable for sensitive high-throughput substrate binding analysis.     

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

The folding mechanism of outer membrane proteins (OMPs) of Gram-negative bacteria into lipid bilayers has been studied using OmpA of E. coli and FomA of F. nucleatum as examples. Both, OmpA and FomA are soluble in unfolded form in urea and insert and fold into phospholipid bilayers upon strong dilution of the denaturant urea. OmpA is a structural protein and forms a small ion channel, composed of an 8-stranded transmembrane beta-barrel domain. FomA is a voltage-dependent porin, predicted to form a 14 stranded beta-barrel. Both OMPs fold into a range of model membranes of very different phospholipid compositions. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers that demonstrated a highly synchronized mechanism of secondary and tertiary structure formation of beta-barrel membrane proteins. A study on FomA folding into lipid bilayers indicated the presence of parallel folding pathways for OMPs with larger transmembrane beta-barrels.     

Does diabetes mellitus affect diphenylhexatriene penetration into erythrocyte membrane ghosts?

Diphenylhexatriene transverse distribution has been studied in normal and diabetic erythrocyte membrane ghosts using fluorescence polarization and fluorescence quenching methods. Acrylamide quenched the fluorescence of diphenylhexatriene according to a dynamic mechanism in agreement with Stern-Volmer equation. Nonlinear least-squares analysis based on quenching results has shown greater accessibility of fluorophore to quencher molecules in diabetic ghosts (37.2 +/- 3.2% in normal vs. 67.5 +/- 6.4% in diabetic membranes). Steady-state fluorescence anisotropy measurements evidenced the lowered membrane lipid fluidity in diabetics (anisotropy values: 0.166 +/- 0.011 in normal subjects vs. 0.193 +/- 0.018 in diabetics). A model mechanism is proposed which attributes the lowered capacity of lipid bilayer in diabetes to the increased ordering and more compact structure of membrane phospholipids. The implications of the results for the resolving of steady-state anisotropy data are discussed.     

Intracellular Ca(2+) release triggers translocation of membrane marker FM1-43 from the extracellular leaflet of plasma membrane into endoplasmic reticulum in T lymphocytes

Stimulation of T cell receptor in lymphocytes enhances Ca(2+) signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1-43 to explore membrane trafficking upon mobilization of intracellular Ca(2+) in Jurkat T cells. We established that liberation of intracellular Ca(2+) with T cell receptor agonist phytohemagglutinin P or with Ca(2+)-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1-43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1-43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1-43-containing ER were free of dye. Intralumenal FM1-43 accumulation was observed even when Ca(2+) signaling in the cytosol was abolished by the removal of extracellular Ca(2+). Our findings strongly suggest that release of intracellular Ca(2+) may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca(2+) elevation.