Lipid Membrane Fusion Research Papers (original) (raw)

Electrical charging of lipid membranes causes electroporation with sharp membrane conductance increases. Several recent observations, especially at very high field strength, are not compatible with the simple electroporation picture. Here we present several relevant experiments on cell electrical responses to very high external voltages. We hypothesize that, not only are aqueous pores created within the lipid membranes, but that nanoscale membrane fragmentation occurs, possibly with micelle formation. This effect would produce conductivity increases beyond simple electroporation and display a relatively fast turn-off with external voltage. In addition, material loss can be expected at the anode side of cells, in agreement with published experimental reports at high fields. Our hypothesis is qualitatively supported by molecular dynamics simulations. Finally, such cellular responses might temporarily inactivate voltage-gated and ion-pump activity, while not necessarily causing cell death. This hypothesis also supports observations on electrofusion.

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- Bioelectrochemistry, Lipid Membrane Fusion, Molecular Dynamics, Computer Simulation

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The 13 C chemical shifts of 13 C, 15 N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/ DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C α and C β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.

- by Su Yongchao
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- Molecular Biology, Kinetics, Magnetic Resonance Spectroscopy, Solid State NMR

Resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. Recent work suggests a critical role for dysferlin in the membrane repair process, and that mutations in dysferlin are responsible for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Beyond membrane repair, dysferlin has been linked to SNARE mediated exocytotic events including cytokine release and acid sphingomyelinase secretion. H o w e ve r , i t i s u n c l e a r w h e t h e r d ys f e r l i n r e gu l a t e s S N A R E m e d i a t e d me m b r a n e f u s i o n. In this study we demonstrate a direct interaction between dysferlin and the SNARE proteins syntaxin 4 and SNAP-23. In addition, analysis of FRET and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates syntaxin 4/SNAP-23 heterodimer formation and SNARE mediated lipid mixing in a calcium sensitive manner. These results support a function for dysferlin as a calcium sensing SNARE effector for membrane fusion events.

- by Colin Johnson
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- Calcium Signaling Pathways, Membrane Trafficking, Lipid Membrane Fusion, SNAREs

Lateral pressure profiles have been suggested to play a significant role in many cellular membrane processes by affecting, for example, the activation of membrane proteins through changes in their conformational state. This may be the case if the lateral pressure profile is altered due to changes in molecular composition surrounding the protein. In this work, we elucidate the effect of varying sterol type on the lateral pressure profile, an issue of topical interest due to lipid rafts and their putative role for membrane protein functionality. We find that the lateral pressure profile is altered when cholesterol is replaced by either desmosterol, 7-dehydrocholesterol, or ketosterol. The observed changes in the lateral pressure profile are notable and important since desmosterol and 7-dehydrocholesterol are the immediate precursors of cholesterol along its biosynthetic pathway. The results show that the lateral pressure profile and the resulting elastic behavior of lipid membranes are sensitive to the sterol type, and support a mechanism where changes in protein conformational state are facilitated by changes in the lateral pressure profile. From a structural point of view, the results provide compelling evidence that despite seemingly minor differences, sterols are characterized by structural specificity.

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membrane fusion and fission events. While initially determined to share many of the features of members of the synaptotagmin family of calcium sensors, ferlins in more recent studies have been found to interact directly with non-neuronal voltage-gated calcium channels and nucleate the assembly of membrane-trafficking protein complexes, functions that distinguish them from the more well studied members of the synaptotagmin family. Here we highlight some of the recent findings that have advanced our understanding of ferlins and their functional differences with the synaptotagmin family.

Lipid membranes, particularly under nonequilibrium conditions, have recently been investigated ever more vigorously because of their relevance in the biological context. We survey our recent approaches to the theoretical study of lipid bilayers that are perturbed in different ways. Self-organization phenomena involving curvature and/or composition spatiotemporal organization are investigated in membrane systems subjected to externally induced chemical reactions, transversal mass transport and insertion of proteins. The outcomes of these studies are expected to be applicable to different curvature and lateral organization phenomena in synthetic lipid bilayers and also in plasmatic cell membranes.

- by Katja Lindenberg
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- Pattern Formation, Lipid Membrane Fusion

Tributyltin is a potent biocide mainly used in marine antifouling paints. Owing to its widespread distribution in coast areas and its high toxicity to aquatic organisms, the use of this compound is generally restricted and under government regulation. Despite of that, it persists in the aquatic environment. Organotins used in industry have also been detected in terrestrial environments. The persistence and high lipophilicity explain bioaccumulation. The role of bacteria in recycling organic matter prompted us to study the interaction of tributyltin with two ubiquitous bacilli, B. stearothermophilus and B. subtilis, proposed as biological indicators of pollutants with ecological impact. These bacteria have been used as suitable models for the study of toxicity mechanisms of unselective lipophilic compounds (e.g., DDT and endosulfan). Drug effects on growth parameters, oxygen consumption and membrane organization were assessed. Bacteria growth in a liquid complex medium was disturbed by concentrations of TBT as low as 25 nM (8 lg L À1 ), close to the concentration in polluted environments. The respiratory activity is affected by TBT in both microorganisms. Membrane organization, assessed by fluorescence polarization of two fluidity probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and a propionic acid derivative (DPH-PA), was also perturbed by the xenobiotic. Alterations on growth, oxygen consumption and physical properties of membrane lipids are stronger in B. stearothermophilus as compared to B. subtilis. A putative relationship between growth inhibition and respiratory activity impairment induced by TBT and its effects on the physical behaviour of bacterial membrane lipids is suggested.

- by Amália Jurado
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- Lipid Membrane Fusion, Lipid Membranes, Propionic Acid, Temperature

31 P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in 31 P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid 31 P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type 31 P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides. D

- by Valerio Parisi
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- Ion Channels, Lipid Membrane Fusion, Biological Sciences, Bacterial Toxins

We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic Nterminal peptide of the HIV-1 LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower. Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the αhelical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the 13 C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains and disrupted the lipid align-ment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities of FPI and that they are worthy of further study.

- by Cyril Curtain
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- Biophysics, Phospholipids, Solid State NMR, Lipid Membrane Fusion

The ability of antibodies binding the influenza hemagglutinin protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A hemagglutinin (HA) protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or Fab fragments bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently-labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA, while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.
DOI: 10.1073/pnas.1411755111

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25-95 μM) and low affinities (Kd = 400-700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B-C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1'-myoinositol-4',5'-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

- by Colin Johnson
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- Membrane Trafficking, Lipid Membrane Fusion, SNAREs, Endocytosis & exocytosis

Lutein is present in the human retina and lens, where it plays a protective role. As lutein is associated with the lipid matrix of biomembranes, the role depends on its membrane location. Experimental studies predicted two orientations of lutein in a phosphatidylcholine (PC) bilayer: vertical and horizontal. Using a molecular dynamics simulation, we observed, in two different PC bilayers, both orientations of lutein, and in each bilayer, a single change from vertical to horizontal orientation or vice versa. Both orientations were stabilized by hydrogen bonding of lutein OH groups with mainly carbonyl but also phosphate oxygen atoms of PC.

Phase diagrams of the hydrated pseudo-binary mixtures dilauroylphosphatidylcholine (DLPC)/lauric acid (LA) and dimyristoylphosphatidylcholine (DMPC)/myristic acid (MA) have been constructed using high-sensitivity DSC and time-resolved X-ray diffraction. They are of a different type compared to those of the longer chain plhosphatidylcholine (PC)/fatty acid (FA) mixtures, the latter being of maximum azeotropic point type. Eutectic points were distinguished in the phase diagrams, at 75 mol'% MA and 49°C for the DMPCjMA mixture, and at ca. 67 mol% LA and 29°C for the DLPC/LA mixture. Regions of liquid&liquid and solid-solid phase separation have been located according to the shape of the phase diagrams and observed by X-ray diffraction.

Recent data depict membranes as the main sites where proteins/peptides are recruited and concentrated, misfold, and nucleate amyloids; at the same time, membranes are considered key triggers of amyloid toxicity. The N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) in 30% trifluoroethanol undergoes a complex path of fibrillation starting with initial 2-3-nm oligomers and culminating with the appearance of mature fibrils. Oligomers are highly cytotoxic and permeabilize lipid membranes, both biological and synthetic. In this article, we report an in-depth study aimed at providing information on the surface activity of HypF-N and its interaction with synthetic membranes of different lipid composition, either in the native conformation or as amyloid oligomers or fibrils. Like other amyloidogenic peptides, the natively folded HypF-N forms stable films at the air/water interface and inserts into synthetic phospholipid bilayers with efficiencies depending on the type of phospholipid. In addition, HypF-N prefibrillar aggregates interact with, insert into, and disassemble supported phospholipid bilayers similarly to other amyloidogenic peptides. These results support the idea that, at least in most cases, early amyloid aggregates of different peptides and proteins produce similar effects on the integrity of membrane assembly and hence on cell viability.

- by Claudio Canale
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- Interaction, Protein, Lipid Membrane Fusion, Amyloid

Recent developments in studies concerning cell-sized vesicles, such as liposomes with a lipid bilayer and water-in-oil droplets covered by a lipid monolayer, aim to realize the real-world modeling of living cells. Compartmentalization with a membrane boundary is essential for the organization of living systems. Due to the relatively large surface/volume ratio in microconfinement, the membrane interface influences phenomena related to biological functions. In this article, we mainly focus on the following subjects: (i) conformational transition of biopolymers in a confined space; (ii) molecular association on the membrane surface; and (iii) remote control of cell-sized membrane morphology.

- by Kenichi Yoshikawa
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- Engineering, Nanoparticle, Materials, Lipid Membrane Fusion

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organel...

- by Sergei Pilyugin
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- Genetics, Lipid Membrane Fusion, Membrane Fusion, Molecular sciences

Principal component analysis (PCA) of time-of-flight secondary ion mass spectrometry (TOF-SIMS) data enables differentiating structurally similar molecules according to linear combinations of multiple peaks in their spectra. However, in order to use PCA to correctly identify variations in lipid composition between samples, the discrimination achieved must be based on chemical differences that are related to the lipid species, and not sample-associated contamination. Here, we identify the positive-ion TOF-SIMS peaks that are related to phosphatidylcholine lipid headgroups and tail groups by PCA of spectra acquired from lipid isotopologs. We demonstrate that restricting PCA to a contaminant-free lipid-related peak set reduces the variability in the spectra acquired from lipid samples that is due to contaminants, which enhanced differentiating different lipid standards, but adversely affected the contrast in PC scores images of phase-separated lipid membranes. We also show that PCA of a restricted data set consisting of the peaks related to lipids and amino acids increases the likelihood that the discrimination of TOF-SIMS data acquired from intact cells is based on differences in the lipids and proteins on the cell surface, and not sample-specific contamination without compromising sample discrimination. We expect that the lipid-related peak database established herein will facilitate interpreting the TOF-SIMS data and PCA results from studies of both model and cellular membranes, and enhance identifying the origins of the peaks that contribute to discriminating different types of cells.

- by Frances Separovic
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- Biophysics, Phospholipids, Solid State NMR, Lipid Membrane Fusion

Microorganisms are very powerful tools for the supply of information about the toxic effects of lipophilic compounds, since an impairment of cell growth usually occurs as a result of perturbations related, in most cases, with the partition of toxicants in membranes. The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify aand b-endosulfan interactions with the membrane possibly related with the insecticide toxicity. Two approaches have been pursued: (a) bacterial growth is followed and the effects of endosulfan isomers determined; (b) biophysical studies with the fluorescent fluidity probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to assess the effects of aand b-endosulfan on the organization of the membrane lipid bilayer. The effects on growth were quantitatively evaluated by determination of growth parameters, namely the lag phase, the specific growth rate and the cell density reached by cultures in the stationary phase. Growth inhibition by a and b-endosulfan dependent on the concentration is diminished or removed by the addition of 2.5 mm Ca 2+ to bacterial cultures. Fluorescence DPH polarization consistently showed opposite effects of Ca 2+ and aand b-endosulfan on the physical state of bacterial polar lipid dispersions. #

- by Amália Jurado
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- Lipid Membrane Fusion, Lipids, Insecticides, In Vitro

- by Amália Jurado
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- Lipid Membrane Fusion, Lipid Membranes, Propionic Acid, Temperature

Methoprene is an insect juvenile growth hormone mimic, commonly used as a pesticide. Although widely used for the control of several pests, toxic effects on organisms of different phyla have been reported. These events triggered studies to clarify the mechanisms of toxicity of this insecticide putatively involved in ecological issues. Here we show the effect of methoprene on the normal cell growth and viability of a strain of the thermophilic eubacterium Bacillus stearothermophilus, previously used as a model for toxicological evaluation of other environment pollutants. Respiration studies were also carried out attempting to identify a putative target for the cytotoxic action of methoprene. Cell growth was affected and a decrease of the number of viable cells was observed as a result of the addition of methoprene to the growth medium, an effect reverted by the presence of Ca 2+ . Methoprene also inhibited the redox flow of B. stearothermophilus protoplasts before the cytochrome oxidase segment, an effect further studied by individually assessing the enzymatic activities of the respiratory complexes. This study suggests that methoprene membrane interaction and perturbation of cell bioenergetics may underlie the mechanism of toxicity of this compound in non-target organisms.

- by Amália Jurado
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- Calcium, Pesticide Residues, Lipid Membrane Fusion, Temperature

Interaction of electric fields with biological systems has begun to receive considerable attention for applications that include field-assisted drug delivery, medical interventions, and genetic engineering. External fields induce the strongest effects at membranes with electroporation being a common feature. Membrane transport in this context of poration is often based on continuum approaches utilizing macroscopic parameters such as the permittivity, diffusion coefficients, and mobilities. In such modeling, field dependences, local inhomogeneities, and microscopic details are usually ignored. Here, a molecular dynamics (MD) scheme is used for a more rigorous and physically realistic evaluation of such parameters for potential application to electroporative transport model development. A suitable membrane structure containing a nanopore derived from MD analysis is used as the initial geometric configuration. Both static and frequency dependent diffusion coefficients have been evaluated. Permittivities are also calculated and shown to be dramatically non-uniform in the vicinity of membranes under high external fields. A positive feedback mechanism leading to enhanced membrane fields is discussed.

We introduce a novel assay for membrane fusion of solid supported membranes on silica beads and on coverslips. Fusion of the lipid bilayers is induced by bringing an optically trapped bead in contact with the coverslip surface while observing the bead's thermal motion with microsecond temporal and nanometer spatial resolution using a three-dimensional position detector. The probability of fusion is controlled by the membrane tension on the particle. We show that the progression of fusion can be monitored by changes in the three-dimensional position histograms of the bead and in its rate of diffusion. We were able to observe all fusion intermediates including transient fusion, formation of a stalk, hemifusion and the completion of a fusion pore. Fusion intermediates are characterized by axial but not lateral confinement of the motion of the bead and independently by the change of its rate of diffusion due to the additional drag from the stalk-like connection between the two membranes. The detailed information provided by this assay makes it ideally suited for studies of early events in pure lipid bilayer fusion or fusion assisted by fusogenic molecules.

- by Tobias Bartsch
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- Biophysics, Optical Tweezers, Membranes, Lipid Membrane Fusion

- by Ranieri Rolandi and +1
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- Biochemistry, Protein Folding, Phospholipids, Interaction

The mechanism of vesicle formation as well as thé précise reasons for their stability are not known. Thus, it is necessary to simulate thé process in vitro for studying its mechanism. If phospholipids are suspendcd in physiological solution by means of cholate and thé détergent is then removcd by dialysis, thé phospholipids sclf-assemble to form unilamcllar vesicles. Wc report hère that thé addition of Na,K-ATPase (an intégral membrane protein) to thé phospholipids changes thé vesicle structure, thcy become larger and a multilamellar population appears. By contrast, carboxyfluorescein, a compound commonly used for labelling thé aqueous vesicle compartment, produccs an unexpectcd effect on vesicle structure by inducing complex, tore-like intravesicular multilayer formations associatcd with a 5-fold increase in diameter. Thus, thc présence of a protein in thé membrane phase or of a compound in thé water phase can influence and direct vesicle formation in vitro; thèse model Systems might give some dues to possible physicochemical or biological factors governing thé formation of natural membrane structures.

The transmembrane domains (TMDs) of membrane-fusogenic proteins contain an overabundance of β-branched residues. In a previous effort to systematically study the relation among valine content, fusogenicity, and helix dynamics, we developed model TMDs that we termed LV-peptides. The content and position of valine in LV-peptides determine their fusogenicity and backbone dynamics, as shown experimentally. Here, we analyze their conformational dynamics and the underlying molecular forces using molecular-dynamics simulations. Our study reveals that backbone dynamics is correlated with the efficiency of side-chain to side-chain van der Waals packing between consecutive turns of the helix. Leu side chains rapidly interconvert between two rotameric states, thus favoring contacts to its i±3 and i±4 neighbors. Stereochemical restraints acting on valine side chains in the α-helix force both β-substituents into an orientation where i,i±3 interactions are less favorable than i,i±4 interactions, thus inducing a local packing deficiency at VV3 motifs. We provide a quantitative molecular model to explain the relationship among chain connectivity, side-chain mobility, and backbone flexibility. We expect that this mechanism also defines the backbone flexibility of natural TMDs.

The lag-burst behavior in the action of phospholipase A 2 (PLA 2 ) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T m of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Fö rster resonance energy transfer between donor-and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA 2 , evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA 2 , involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form ''mature'' fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.

- by christian code
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- Interaction, Protein, Lipid Membrane Fusion, Amyloid