Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix

The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins.     

Oxonol VI as an optical indicator for membrane potentials in lipid vesicles

Experiments with large unilamellar dioleoylphosphatidylcholine vesicles were carried out in order to study the effect of membrane potential on the fluorescence of Oxonol VI. A partition equilibrium of dye between membrane and water was found to exist with a partition coefficient gamma identical to c lipid/c water of about 19,000 (at zero voltage). In the presence of an inside-positive membrane potential, the negatively charged dye accumulates in the intravesicular aqueous space according to a Nernst equilibrium. This leads to an increased adsorption of dye to the inner lipid monolayer and to a concomitant increase of fluorescence. The fluorescence change can be calibrated as a function of transmembrane voltage by generating a potassium diffusion potential in the presence of valinomycin. The intrinsic fluorescence of the membrane-bound dye is not affected by voltage; the whole influence of voltage on the fluorescence results from voltage-dependent partitioning of the dye between water and membrane. The voltage dependence of the apparent partition coefficient can be quantitatively described by a three-capacitor model in which the dye is assumed to bind to adsorption planes located on the hydrocarbon side of the membrane/solution interface. Oxonol VI was found to be suitable for detecting changes of membrane potential associated with the activity of the (Na+ + K+)-ATPase in reconstituted vesicles. When ATP is added to the external medium, pump molecules with the ATP-binding side facing outward become activated; this results in a translocation of net positive charge towards the vesicle interior. Under this condition, fluorescence changes corresponding to (inside-positive) potentials of up to 150-200 mV are observed. After the build-up of the membrane potential, a quasi-stationary state is reached in which the pump current is compensated by a back-flow of charge through passive conductance pathways.     

Surface potential on purple membranes and its sidedness studied by a resonance Raman dye probe.

A new technique for the measurement of membrane surface potential is proposed and demonstrated. The method is based on the fact that a positively charged styryl dye molecule aggregates when present at high concentration in the Debye layer near a membrane bearing a negative surface potential. The dye in its aggregated form exhibits marked differences in its resonance Raman spectrum relative to the free dye molecules. This method was used to study the potential on the surfaces of the purple membrane that contains the pigment bacteriorhodopsin. A value of -29.5 mV was found for membranes with bacteriorhodopsin in its relaxed, light-adapted state, and the potential decreased to -34.5 mV when most of the bacteriorhodopsin was converted to the M412 intermediate. Because the dye probe does not diffuse through the lipid bilayer, it can be used to probe the potential on the external or internal surface of a vesicle. Thus, we found that the potential on the purple membrane was asymmetric and was localized mainly on the surface that faces the cytoplasm in the cell.     

Coarse-grained molecular dynamics simulations of the energetics of helix insertion into a lipid bilayer

Experimental and computational studies have indicated that hydrophobicity plays a key role in driving the insertion of transmembrane alpha-helices into lipid bilayers. Molecular dynamics simulations allow exploration of the nature of the interactions of transmembrane alpha-helices with their lipid bilayer environment. In particular, coarse-grained simulations have considerable potential for studying many aspects of membrane proteins, ranging from their self-assembly to the relation between their structure and function. However, there is a need to evaluate the accuracy of coarse-grained estimates of the energetics of transmembrane helix insertion. Here, three levels of complexity of model system have been explored to enable such an evaluation. First, calculated free energies of partitioning of amino acid side chains between water and alkane yielded an excellent correlation with experiment. Second, free energy profiles for transfer of amino acid side chains along the normal to a phosphatidylcholine bilayer were in good agreement with experimental and atomistic simulation studies. Third, estimation of the free energy profile for transfer of an arginine residue, embedded within a hydrophobic alpha-helix, to the center of a lipid bilayer gave a barrier of approximately 15 kT. Hence, there is a substantial barrier to membrane insertion for charged amino acids, but the coarse-grained model still underestimates the corresponding free energy estimate (approximately 29 kT) from atomistic simulations (Dorairaj, S., and Allen, T. W. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 4943-4948). Coarse-grained simulations were then used to predict the free energy profile for transfer of a simple model transmembrane alpha-helix (WALP23) across a lipid bilayer. The results indicated that a transmembrane orientation was favored by about -70 kT.     

Rotational dynamics of phospholamban determined by multifrequency electron paramagnetic resonance

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics (T) and another with nearly unrestricted subnanosecond motion (R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

Hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.     

Dynamics of the Kv1.2 voltage-gated K+ channel in a membrane environment

All-atom molecular dynamics simulations are used to better understand the dynamic environment experienced by the Kv1.2 channel in a lipid membrane. The structure of the channel is stable during the trajectories. The pore domain keeps a well-defined conformation, whereas the voltage-sensing domains undergo important lateral fluctuations, consistent with their modular nature. A channel-like region at the center of the S1-S4 helical bundle fills rapidly with water, reminiscent of the concept of high-dielectric aqueous crevices. The first two arginines along S4 (R294 and R297) adopt an interfacial position where they interact favorably with water and the lipid headgroups. The following two arginines (R300 and R303) interact predominantly with water and E226 in S2. Despite the absence of a structurally permanent gating pore formed by protein residues and surrounding the S4 helix, as traditionally pictured, the charged residues are located in a favorable environment and are not extensively exposed to the membrane nonpolar region. Continuum electrostatic computations indicate that the transmembrane potential sensed by the charged residues in the voltage sensor varies abruptly over the outer half of the membrane in the arginine-rich region of S4; thus, the voltage gradient or membrane electric field is "focused". Interactions of basic residues with the lipid headgroups at the intracellular membrane-solution interface reduce the membrane thickness near the channel, resulting in an increased transmembrane field.     

Signal transduction by CD58: The transmembrane isoform transmits signals outside lipid rafts independently of the GPI-anchored isoform

The adhesion molecule CD58 is natively expressed in both a glycosylphosphatidylinositol (GPI)-anchored form and a transmembrane form. We previously demonstrated that the two isoforms of CD58 are differentially distributed in the cell membrane. The GPI-linked form resides in lipid rafts while the transmembrane form resides outside lipid rafts. Following cross-linking a fraction of transmembrane CD58 redistributes to lipid rafts. It has also been demonstrated that ligand binding to CD58 induces biological functions such as cytokine production and immunoglobulin isotype switching, indicating that cell–cell interactions result in CD58-mediated signal transduction. However, the signaling pathways involved in these activation processes are poorly defined. Here we show for the first time that cross-linking of CD58 induces protein tyrosine phosphorylation of BLNK, Syk and PLCγ, and activation of ERK and Akt/PKB. In addition, we studied how these signaling events relate to the distinct membrane localization of the two isoforms of CD58. We demonstrate that cross-linking of CD58 triggers signaling that is predominantly associated with transmembrane CD58 in nonraft microdomains. Moreover, signaling through transmembrane CD58 does not depend on coexpression of the GPI-linked isoform. Thus, despite the residence of its GPI-anchored isoform in lipid rafts and the translocation of a fraction of its transmembrane isoform to lipid rafts, CD58 signaling is triggered by the transmembrane isoform outside lipid rafts. These findings corroborate signaling outside lipid rafts, as opposed to the established notion that rafts function as essential platforms for signaling.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

The lipid environment of the nicotinic acetylcholine receptor in native and reconstituted membranes

Detailed knowledge of the membrane framework surrounding the nicotinic acetylcholine receptor (AChR) is key to an understanding of its structure, dynamics, and function. Recent theoretical models discuss the structural relationship between the AChR and the lipid bilayer. Independent experimental data on the composition, metabolism, and dynamics of the AChR lipid environment are analyzed in the first part of the review. The composition of the lipids in which the transmembrane AChR chains are inserted bears considerable resemblance among species, perhaps providing this evolutionarily conserved protein with an adequate milieu for its optimal functioning. The effects of lipids on the latter are discussed in the second part of the review. The third part focuses on the information gained on the dynamics of AChR and lipids in the membrane, a section that also covers the physical properties and interactions between the protein, its immediate annulus, and the bulk lipid bilayer.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

Structure and Orientation of the Mammalian Antibacterial Peptide Cecropin P1 within Phospholipid Membranes

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly α-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospho lipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to acispositive transmembrane voltage difference. Taken together, the results further support a “carpet-like” mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.     

Sensitivity of erythrocyte acethylcholinesterase to inhibition by linolenoyl sorbitol. Dependence on a transmembrane potential.

Acetylcholinesterase activity of human erythrocytes is known to be inhibited by linolenoyl sorbitol, the inhibition being critically dependent on cell membrane intactness. The extent of enzyme inhibition by the added lipid is correlated with the magnitude of Cl- gradient across the erythrocyte membrane, indicating that enzyme sensitivity is associated with a transmembrane potential. If linolenoyl sorbitol is allowed to interact with the erythrocytes while a Cl- gradient exists, enzyme sensitivity can subsequently be demonstrated not only in the absence of a gradient but even when the cells are lyzed. It is consluded that the transmembrane potential determines the accessibility of a membrane component to the added lipid.     

Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning.     

Bilayer mechanical properties regulate the transmembrane helix mobility and enzymatic state of CD39

CD39 can exist in at least two distinct functional states depending on the presence and intact membrane integration of its two transmembrane helices. In native membranes, the transmembrane helices undergo dynamic rotational motions that are required for enzymatic activity and are regulated by substrate binding. In this study, we show that bilayer mechanical properties regulate conversion between the two enzymatic functional states by modulating transmembrane helix dynamics. Alteration of membrane properties by insertion of cone-shaped or inverse cone-shaped amphiphiles or by cholesterol removal switches CD39 to the same enzymatic state that removal or solubilization of the transmembrane domains does. The same membrane alterations increase the propensity of both transmembrane helices to rotate within the packed structure, resulting in a structure with greater mobility but not an altered primary conformation. Membrane alteration also abolishes the ability of the substrate to stabilize the helices in their primary conformation, indicating a loss of coupling between substrate binding and transmembrane helix dynamics. Removal of either transmembrane helix mimics the effect of membrane alteration on the mobility and substrate sensitivity of the remaining helix, suggesting that the ends of the extracellular domain have intrinsic flexibility. We suggest that a mechanical bilayer property, potentially elasticity, regulates CD39 by altering the balance between the stability and flexibility of its transmembrane helices and, in turn, of its active site.     

Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin

During vertebrate development, oligodendrocytes wrap their plasma membrane around axons to produce myelin, a specialized membrane highly enriched in galactosylceramide (GalC) and cholesterol. Here, we studied the formation of myelin membrane sheets in a neuron-glia co-culture system. We applied different microscopy techniques to visualize lipid packing and dynamics in the oligodendroglial plasma membrane. We used the fluorescent dye Laurdan to examine the lipid order with two-photon microscopy and observed that neurons induce a dramatic lipid condensation of the oligodendroglial membrane. On a nanoscale resolution, using stimulated emission depletion and fluorescence resonance energy transfer microscopy, we demonstrated a neuronal-dependent clustering of GalC in oligodendrocytes. Most importantly these changes in lipid organization of the oligodendroglial plasma membrane were not observed in shiverer mice that do not express the myelin basic protein. Our data demonstrate that neurons induce the condensation of the myelin-forming bilayer in oligodendrocytes and that MBP is involved in this process of plasma membrane rearrangement. We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction.     

Transmembrane peptides from tyrosine kinase receptor. Mutation-related behavior in a lipid bilayer investigated by molecular dynamics simulations

Polar mutations in transmembrane alpha helices may alter the structural details of the hydrophobic sequences and control intermolecular contacts. We have performed molecular dynamics simulations on the transmembrane domain of the proto-oncogenic and the oncogenic forms of the Neu receptor in a fluid DMPC bilayer to test whether the Glu mutation which replaces the Val residue at position 664 may alter the helical structure and its insertion in the membrane. The simulations show that the wild and the mutant forms of the transmembrane domain have a different behavior in the bilayer. The native transmembrane sequence is found to be more flexible than in the presence of the Glu mutation, characterized by a tendency to pi deformation to accommodate the helix length to the membrane thickness. The mutant form of this domain does not evidence helical deformation in the present simulation. Hydrophobic matching is achieved both by a larger helix tilt and a vertical shift of the helix towards the membrane interface, favoring the accessibility of the Glu side chain to the membrane environment. A rapid exchange of hydrogen bond interactions with the surrounding water molecules and the lipid headgroups is observed. The difference in the behavior between the two peptides in a membrane environment was also observed experimentally. Both simulation and experimental results agree with the hypothesis that water may act as an intermediate for the formation of cross links between the facing Glu side chains stabilizing the dimer.     

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.