Attenuated total reflection IR spectroscopy as a tool to investigate the orientation and tertiary structure changes in fusion proteins

Membrane fusion proceeds via a merging of two lipid bilayers and a redistribution of aqueous contents and bilayer components. It involves transition states in which the phospholipids are not arranged in bilayers and in which the monolayers are highly curved. Such transition states are energetically unfavourable since biological membranes are submitted to strong repulsive hydration electrostatic and steric barriers. Viral membrane proteins can help to overcome these barriers. Viral proteins involved in membrane fusion are membrane associated and the presence of lipids restricts drastically the potential of methods (RMN, X-ray crystallography) that have been used successfully to determine the tertiary structure of soluble proteins. We describe here how IR spectroscopy allows to solve some of the problems related to the lipid environment.The principles of the method, the experimental setup and the preparation of the samples are briefly described. A few examples illustrate how attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy can be used to gain information on the orientation and the accessibility to the water phase of the fusogenic domain of viral proteins. Recent developments suggest that the method could also be used to detect changes located in the membrane domains and to identify intermediate structural states involved in the fusion process.     

Adhesion and merging of lipid bilayers: a method for measuring the free energy of adhesion and hemifusion

Lipid bilayers can be induced to adhere to each other by molecular mediators, and, depending on the lipid composition, such adhesion can lead to merging of the contacting monolayers in a process known as hemifusion. Such bilayer-bilayer reactions have never been systematically studied. In the course of our studies of membrane-active molecules, we encountered such reactions. We believe that they need to be understood whenever bilayer-bilayer interactions take place, such as during membrane fusion. For illustration, we discuss three examples: spontaneous adhesion between phospholipid bilayers induced by low pH, polymer-induced osmotic depletion attraction between lipid bilayers, and anionic lipid bilayers cross-bridged by multicationic peptides. Our purpose here is to describe a general method for studying such interactions. We used giant unilamellar vesicles, each of which was aspirated in a micropipette so that we could monitor the tension of the membrane and the membrane area changes during the bilayer-bilayer interaction. We devised a general method for measuring the free energy of adhesion or hemifusion. The results show that the energies of adhesion or hemifusion of lipid bilayers could vary over 2 orders of magnitude from −1 to −50 × 10⁻⁵ J/m² in these examples alone. Our method can be used to measure the energy of transition in each step of lipid transformation during membrane fusion. This is relevant for current research on membrane fusion, which focuses on how fusion proteins induce lipid transformations.     

Studying membrane fusion using supported lipid bilayers on superparamagnetic beads

The fusion between two lipid membranes is a ubiquitous mechanism in cell traffic and pathogens invasion. Yet it is not well understood how two distinct bilayers overcome the energy barriers towards fusion and reorganize themselves to form a unique continuous bilayer. The magnitudes and numbers of these energy barriers are themselves an open question. To tackle these issues, we developed a new tool that allows to control the forces applied between two supported lipid bilayers (SLBs) deposited on superparamagnetic beads. By applying a magnetic field, the beads self-organize along field lines in chains of beads and compress the two membranes on the contact zone. Using the diffusion of fluorescently labelled lipids from one bilayer to the other allows us to identify fusion of the bilayers in contact. We applied increasing forces on SLBs and increased the occurrence of fusion. This experimental system allows the simultaneous study of tens of facing bilayers in a single experiment and mitigates the stochasticity of the fusion process. It is thus a powerful tool to test the various parameters involved in the membrane fusion process.     

Single-Molecule Force Spectroscopy of Membrane Protein Folding

Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Quantification of phase transitions of lipid mixtures from bilayer to non-bilayer structures: Model,experimental validation and implication on membrane fusion

Lipid bilayers provide a solute-proof barrier that is widely used in living systems. It has long been recognized that the structural changes of lipids during the phase transition from bilayer to non-bilayer have striking similarities with those accompanying membrane fusion processes. In spite of this resemblance, the numerous quantitative studies on pure lipid bilayers are difficult to apply to real membranes. One reason is that in living matter, instead of pure lipids, lipid mixtures are involved and there is currently no model that establishes the connection between pure lipids and lipid mixtures. Here, we make this connection by showing how to obtain (i) the short-range repulsion between bilayers made of lipid mixtures and, (ii) the pressure at which transition from bilayer phase to non-bilayer phases occur. We validated our models by fitting the experimental data of several lipid mixtures to the theoretical data calculated based on our model. These results provide a useful tool to quantitatively predict the behavior of complex membranes at low hydration.     

Insertion of lipid domains into plasma membranes by fusion with erythrocytes

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.     

Calculating Transition Energy Barriers and Characterizing Activation States for Steps of Fusion

We use continuum mechanics to calculate an entire least energy pathway of membrane fusion, from stalk formation, to pore creation, and through fusion pore enlargement. The model assumes that each structure in the pathway is axially symmetric. The static continuum stalk structure agrees quantitatively with experimental stalk architecture. Calculations show that in a stalk, the distal monolayer is stretched and the stored stretching energy is significantly less than the tilt energy of an unstretched distal monolayer. The string method is used to determine the energy of the transition barriers that separate intermediate states and the dynamics of two bilayers as they pass through them. Hemifusion requires a small amount of energy independently of lipid composition, while direct transition from a stalk to a fusion pore without a hemifusion intermediate is highly improbable. Hemifusion diaphragm expansion is spontaneous for distal monolayers containing at least two lipid components, given sufficiently negative diaphragm spontaneous curvature. Conversely, diaphragms formed from single-component distal monolayers do not expand without the continual injection of energy. We identify a diaphragm radius, below which central pore expansion is spontaneous. For larger diaphragms, prior studies have shown that pore expansion is not axisymmetric, and here our calculations supply an upper bound for the energy of the barrier against pore formation. The major energy-requiring deformations in the steps of fusion are: widening of a hydrophobic fissure in bilayers for stalk formation, splay within the expanding hemifusion diaphragm, and fissure widening initiating pore formation in a hemifusion diaphragm.     

Adhesion and hemifusion of cytoplasmic myelin lipid membranes are highly dependent on the lipid composition

We report the effects of calcium ions on the adhesion and hemifusion mechanisms of model supported myelin lipid bilayer membranes of differing lipid composition. As in our previous studies Min et al. [1,2], the lipid compositions used mimic "healthy" and "diseased-like" (experimental autoimmune encephalomyelitis, EAE) membranes. Our results show that the interaction forces as a function of membrane separation distance are well described by a generic model that also (and in particular) includes the hydrophobic interaction arising from the hydrophobically exposed (interior) parts of the bilayers. The model is able to capture the mechanical instability that triggers the onset of the hemifusion event, and highlights the primary role of the hydrophobic interaction in membrane fusion. The effects of lipid composition on the fusion mechanism, and the adhesion forces between myelin lipid bilayers, can be summarized as follows: in calcium-free buffer, healthy membranes do not present any signs of adhesion or hemifusion, while diseased membranes hemifuse easily. Addition of 2mM calcium favors adhesion and hemifusion of the membranes independently of their composition, but the mechanisms involved in the two processes were different: healthy bilayers systematically presented stronger adhesion forces and lower energy barriers to fusion compared to diseased bilayers. These results are of particular relevance for understanding lesion development (demyelination, swelling, vacuolization and/or vesiculation) in myelin associated diseases such as multiple sclerosis and its relationship to lipid domain formation in myelin membranes.     

Individual Vesicle Fusion Events Mediated by Lipid-Anchored DNA

Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.     

Thermodynamics of fusion peptide-membrane interactions

The fusion peptides of viral membrane fusion proteins play a key role in the mechanism of viral spike glycoprotein mediated membrane fusion. These peptides insert into the lipid bilayers of cellular target membranes where they adopt mostly helical secondary structures. To better understand how membranes may be converted to high-energy intermediates during fusion, it is of interest to know how much energy, enthalpy and entropy, is provided by the insertion of fusion peptides into lipid bilayers. Here, we describe a detailed thermodynamic analysis of the binding of analogues of the influenza hemagglutinin fusion peptide of different lengths and amino acid compositions. In small unilamellar vesicles, the interaction of these peptides with lipid bilayers is driven by enthalpy (-16.5 kcal/mol) and opposed by entropy (-30 cal mol(-1) K(-1)). Most of the driving force (deltaG = -7.6 kcal/mol) comes from the enthalpy of peptide insertion deep into the lipid bilayer. Enthalpic gains and entropic losses of peptide folding in the lipid bilayer cancel to a large extent and account for only about 40% of the total binding free energy. The major folding event occurs in the N-terminal segment of the fusion peptide. The C-terminal segment mainly serves to drive the N-terminus deep into the membrane. The fusion-defective mutations G1S, which causes hemifusion, and particularly G1V, which blocks fusion, have major structural and thermodynamic consequences on the insertion of fusion peptides into lipid bilayers. The magnitudes of the enthalpies and entropies of binding of these mutant peptides are reduced, their helix contents are reduced, but their energies of self-association at the membrane surface are increased compared to the wild-type fusion peptide.     

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.     

Resolving the kinetics of lipid, protein and peptide diffusion in membranes

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.     

HIV-1 fusion peptide decreases bending energy and promotes curved fusion intermediates

A crucial step in human immunodeficiency virus (HIV) infection is fusion between the viral envelope and the T-cell membrane, which must involve intermediate membrane states with high curvature. Our main result from diffuse x-ray scattering is that the bending modulus K(C) is greatly reduced upon addition of the HIV fusion peptide FP-23 to lipid bilayers. A smaller bending modulus reduces the free energy barriers required to achieve and pass through the highly curved intermediate states and thereby facilitates fusion and HIV infection. The reduction in K(C) is by a factor of 13 for the thicker, stiffer 1,2-sn-dierucoylphosphatidylcholine bilayers and by a factor of 3 for 1,2-sn-dioleoylphosphatidylcholine bilayers. The reduction in K(C) decays exponentially with concentration of FP-23, and the 1/e concentration is <1 mol % peptide/lipid, which is well within the physiological range for a fusion site. A secondary result is, when FP-23 is added to the samples which consist of stacks of membranes, that the distance between membranes increases and eventually becomes infinite at full hydration (unbinding); we attribute this both to electrostatic repulsion of the positively charged arginine in the FP-23 and to an increase in the repulsive fluctuation interaction brought about by the smaller K(C). Although this latter interaction works against membrane fusion, our results show that the energy that it requires of the fusion protein machinery to bring the HIV envelope membrane and the target T-cell membrane into close contact is negligible.     

Lipid Tail Protrusion in Simulations Predicts Fusogenic Activity of Influenza Fusion Peptide Mutants and Conformational Models

Fusion peptides from influenza hemagglutinin act on membranes to promote membrane fusion, but the mechanism by which they do so remains unknown. Recent theoretical work has suggested that contact of protruding lipid tails may be an important feature of the transition state for membrane fusion. If this is so, then influenza fusion peptides would be expected to promote tail protrusion in proportion to the ability of the corresponding full-length hemagglutinin to drive lipid mixing in fusion assays. We have performed molecular dynamics simulations of influenza fusion peptides in lipid bilayers, comparing the X-31 influenza strain against a series of N-terminal mutants. As hypothesized, the probability of lipid tail protrusion correlates well with the lipid mixing rate induced by each mutant. This supports the conclusion that tail protrusion is important to the transition state for fusion. Furthermore, it suggests that tail protrusion can be used to examine how fusion peptides might interact with membranes to promote fusion. Previous models for native influenza fusion peptide structure in membranes include a kinked helix, a straight helix, and a helical hairpin. Our simulations visit each of these conformations. Thus, the free energy differences between each are likely low enough that specifics of the membrane environment and peptide construct may be sufficient to modulate the equilibrium between them. However, the kinked helix promotes lipid tail protrusion in our simulations much more strongly than the other two structures. We therefore predict that the kinked helix is the most fusogenic of these three conformations.     

Atomic-Resolution Simulations Predict a Transition State for Vesicle Fusion Defined by Contact of a Few Lipid Tails

Membrane fusion is essential to both cellular vesicle trafficking and infection by enveloped viruses. While the fusion protein assemblies that catalyze fusion are readily identifiable, the specific activities of the proteins involved and nature of the membrane changes they induce remain unknown. Here, we use many atomic-resolution simulations of vesicle fusion to examine the molecular mechanisms for fusion in detail. We employ committor analysis for these million-atom vesicle fusion simulations to identify a transition state for fusion stalk formation. In our simulations, this transition state occurs when the bulk properties of each lipid bilayer remain in a lamellar state but a few hydrophobic tails bulge into the hydrophilic interface layer and make contact to nucleate a stalk. Additional simulations of influenza fusion peptides in lipid bilayers show that the peptides promote similar local protrusion of lipid tails. Comparing these two sets of simulations, we obtain a common set of structural changes between the transition state for stalk formation and the local environment of peptides known to catalyze fusion. Our results thus suggest that the specific molecular properties of individual lipids are highly important to vesicle fusion and yield an explicit structural model that could help explain the mechanism of catalysis by fusion proteins.     

Resolving the kinetics of lipid,protein and peptide diffusion in membranes

Abstract

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.     

A solid-state NMR study of changes in lipid phase induced by membrane-fusogenic LV-peptides

Membrane fusion requires restructuring of lipid bilayers mediated by fusogenic membrane proteins. Peptides that correspond to natural transmembrane sequences or that have been designed to mimic them, such as low-complexity “Leu-Val” (LV) peptide sequences, can drive membrane fusion, presumably by disturbing the lipid bilayer structure. Here, we assess how peptides of different fusogenicity affect membrane structure using solid state NMR techniques. We find that the more fusogenic variants induce an unaligned lipid phase component and a large degree of phase separation as observed in ³¹P 2D spectra. The data support the idea that fusogenic peptides accumulate PE in a non-bilayer phase which may be critical for the induction of fusion.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

The Interaction between Influenza HA Fusion Peptide and Transmembrane Domain Affects Membrane Structure

Viral glycoproteins, such as influenza hemagglutinin (HA) and human immunodeficiency virus gp41, are anchored by a single helical segment transmembrane domain (TMD) on the viral envelope membrane. The fusion peptides (FP) of the glycoproteins insert into the host membrane and initiate membrane fusion. Our previous study showed that the FP or TMD alone perturbs membrane structure. Interaction between the influenza HA FP and TMD has previously been shown, but its role is unclear. We used PC spin labels dipalmitoylphospatidyl-tempo-choline (on the headgroup), 5PC and 14PC (5-C and 14-C positions on the acyl chain) to detect the combined effect of FP-TMD interaction by titrating HA FP to TMD-reconstituted 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol)/cholesterol lipid bilayers using electron spin resonance. We found that the FP-TMD increases the lipid order at all positions, which has a greater lipid ordering effect than the sum of the FP or TMD alone, and this effect reaches deeper into the membranes. Although HA-mediated membrane fusion is pH dependent, this combined effect is observed at both pH 5 and pH 7. In addition to increasing lipid order, multiple components are found for 5PC at increased concentration of FP-TMD, indicating that distinct domains are induced. However, the mutation of Gly1 in the FP and L187 in the TMD eliminates the perturbations, consistent with their fusogenic phenotypes. Electron spin resonance on spin-labeled peptides confirms these observations. We suggest that this interaction may provide a driving force in different stages of membrane fusion: initialization, transition from hemifusion stalk to transmembrane contact, and fusion pore formation.