Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.     

The behavior of R-ovalbumin and its individual components A1, A2, and A3 in urea solution: kinetics and equilibria

Procedures are described for the isolation of the individual components A₁, A₂, and A₃ of native R-ovalbumin from freshly laid domestic hen eggs. Because heavy metal ion contaminants result in spurious irreproducible kinetics, particularly at high pH, considerable care is taken to avoid their presence. Kinetics studies are made of the behavior of whole R-ovalbumin and its individual components in urea solution over the pH range 3.7–9.6 following the reaction by determining absorbance differences at 233, 287, and 293 nm and ORD and CD changes at 350 and 221 nm, respectively. Reaction is rapid at low pH, slowing with increasing pH. Except under limited conditions, the reaction is not simple first order. Equations are presented for describing the reactions, and the nature of the reaction products is considered. Unfolding equilibrium profiles were also determined by ORD at several wavelengths and were not stigmoidal in shape and the normalized curves were not superimposed.Deceased December 8, 2001     

Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

Background

The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to α-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative α-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB.

Methodology/Principal Findings

14 α-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 α-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative α-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for α-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved α-helical coiled coil motifs.

Conclusion/Significance

We conclude that the selection of α-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.     

Urea perturbation and the reversibility of nucleohistone conformation

Urea effect on conformation and thermal stabilities in nucleohistone and NaCl-treated partially dehistonized nucleohistones has been studied by circular dichroism (CD) and thermal denaturation. Urea imposes a CD change at 278mm of DNA base pairs in native and NaCl-treated nucleohistones which can be decomposed into two parts: a decrease in Δε₂₇₈ for histone-free base pairs and an increase for histone-bound base pairs. The reduction by urea of Δε₂₂₀ of bound histones is approximately proportional to the increase of Δε₂₇₈ of histone-bound base pairs. Urea also lowers the melting temperatures of base pairs both free and bound by histones. The presence of urea indeed destroys the secondary structure of bound histones, causing changes in the conformation and thermal stabilities of histone-bound base pairs in nucleohistone. Such a urea perturbation on nucleohistone conformation is reversible.     

Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces

Amphipathic α-helices mediate binding of exchangeable apolipoproteins to lipoproteins. To probe the role of α-helical structure in protein-lipid interactions, we used oil-drop tensiometry to characterize the interfacial behavior of apolipoprotein C-I (apoC-I) variants at triolein/water (TO/W) and 1-palmitoyl-2-oleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. ApoC-I, the smallest apolipoprotein, has two amphipathic α-helices. Mutants had single Pro or Ala substitutions that resulted in large differences in helical content in solution and on phospholipids. The ability of apoC-I to bind TO/W and POPC/TO/W interfaces correlated strongly with α-helical propensity. On binding these interfaces, peptides with higher helical propensity increased surface pressure to a greater extent. Likewise, peptide exclusion pressure at POPC/TO/W interfaces increased with greater helical propensity. ApoC-I retention on TO/W and POPC/TO/W interfaces correlated strongly with phospholipid-bound helical content. On compression of these interfaces, peptides with higher helical content were ejected at higher pressures. Substitution of Arg for Pro in the N-terminal α-helix altered net charge and reduced apoC-I affinity for POPC/TO/W interfaces. Our results suggest that peptide-lipid interactions drive α-helix binding to and retention on lipoproteins. Point mutations in small apolipoproteins could significantly change α-helical propensity or charge, thereby disrupting protein-lipid interactions and preventing the proteins from regulating lipoprotein catabolism at high surface pressures.     

Activation of Integrins by Urea in Perfused Rat Liver

High concentrations of urea were shown to induce a paradoxical regulatory volume decrease response with K⁺ channel opening and subsequent hepatocyte shrinkage (Hallbrucker, C., vom Dahl, S., Ritter, M., Lang, F., and Häussinger, D. (1994) Pflügers Arch. 428, 552–560), although the hepatocyte plasma membrane is thought to be freely permeable to urea. The underlying mechanisms remained unclear. As shown in the present study, urea (100 mmol/liter) induced within 1 min an activation of β₁ integrins followed by an activation of focal adhesion kinase, c-Src, p38^MAPK, extracellular signal-regulated kinases, and c-Jun N-terminal kinase. Because α₅β₁ integrin is known to act as a volume/osmosensor in hepatocytes, which becomes activated in response to hepatocyte swelling, the findings suggest that urea at high concentrations induces a nonosmotic activating perturbation of this osmosensor, thereby triggering a volume regulatory K⁺ efflux. In line with this, similar to hypo-osmotic hepatocyte swelling, urea induced an inhibition of hepatic proteolysis, which was sensitive to p38^MAPK inhibition. Molecular dynamics simulations of a three-dimensional model of the ectodomain of α₅β₁ integrin in water, urea, or thiourea solutions revealed significant conformational changes of α₅β₁ integrin in urea and thiourea solutions, in contrast to the simulation of α₅β₁ in water. These changes lead to an unbending of the integrin structure around the genu, which may suggest activation, whereas the structures of single domains remained essentially unchanged. It is concluded that urea at high concentrations affects hepatic metabolism through direct activation of the α₅β₁ integrin system.     

How Natalizumab Binds and Antagonizes α4 Integrins

Natalizumab antibody to α₄-integrins is used in therapy of multiple sclerosis and Crohn''s disease. A crystal structure of the Fab bound to an α₄ integrin β-propeller and thigh domain fragment shows that natalizumab recognizes human-mouse differences on the circumference of the β-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the β-subunit βI domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface α₄β₁ shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to α₄-integrins is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to α₄β₁. Mapping of species-specific differences onto α₄β₁ and α₄β₇ shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.     

NMR structure of the water soluble Aβ17–34 peptide

Alzheimer''s disease is the most common neurodegenerative disorder in the world. Its most significant symptoms are memory loss and decrease in cognition. Alzheimer''s disease is characterized by aggregation of two proteins in the brain namely Aβ (amyloid β) and tau. Recent evidence suggests that the interaction of soluble Aβ with nAChR (nicotinic acetylcholine receptors) contributes to disease progression. In this study, we determine the NMR structure of an Aβ_17–34 peptide solubilized by the addition of two glutamic acids at each terminus. Our results indicate that the Aβ peptide adopts an α-helical structure for residues 19–26 and 28–33. The α-helical structure is broken around residues S26, N27 and K28, which form a kink in the helical conformation. This α-helix was not described earlier in an aqueous solution without organic solvents, and at physiological conditions (pH 7). These data are in agreement with Aβ adopting an α-helical conformation in the membrane before polymerizing into amyloid β-sheets and provide insight into the intermediate state of Aβ in Alzheimer''s disease.     

β-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbβ3 Headpiece

The platelet integrin α_IIbβ₃ binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and β-subunits; the basic Lys or Arg side chain hydrogen bonds to the α_IIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the β₃-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket toward α_IIb, with movement of the βI-domain β1-α1 loop toward α_IIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and α_IIb. Here we test whether ligand interactions with β₃ suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the α_IIbβ₃ headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed α_IIbβ₃ headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the β₃-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the α_IIb subunit on Arg is required.     

Cannabinoid receptor interacting protein 1a interacts with myristoylated Gαi N terminus via a unique gapped β-barrel structure

Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB₁ cannabinoid receptor G-protein coupling in part by altering the selectivity for Gα_i subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel β-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The β-barrel has a gap between strands β8 and β10, which deviates from β-sandwich fatty acid–binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the β-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing β-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gα_i N terminus. However, CRIP1a could not bind the nonmyristolyated Gα_i peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB₁ receptor.     

Characteristics of an R-Phycoerythrin with Two γ Subunits Prepared from Red Macroalga Polysiphonia urceolata

An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α^18.2, β^20.6, γ^31.6 (γ''), γ^34.6 (γ), and no colorless polypeptide; its subunit composition was 6α^18.2:6β^20.6:1 γ^31.6:2γ^34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)₃ γ (αβ)₃γ'' and γ (αβ)₃ γ''(αβ)₃ γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)₃ face-to-face with the aid of one γ subunit (γ or γ'').     

Evidence for unbenignant nature of glucose as a replacement for water in purple membranes

The net angle (θ_α) between the seven helical segments of the bacteriorhodopsin (bR) polypeptide and the normal to the membrane plane of the purple membrane (PM) is ~0° when determined by oriented far-ultraviolet (UV) circular dichroism (OCD) and midinfrared linear dichroism (IRLD). However, θ_α is ~11° when determined by high-resolution electron cryo-microscopy and electron diffraction (EMD). The spectral studies are made with fresh hydrated PM films at ambient temperature, whereas diffraction studies are made with aged glucose-embedded PM at -120 to -268°. The current study presents oriented far-UV OCD results of hydrated PM films embedded with glucose, which can best be interpreted as a change in the magnitude of θ_α (Δθ_α) from 0 to 23° as a consequence of glucose embedment. Possible alternative explanations contrary to this conclusion are discussed and ruled out. Therefore, it is suggested that a θ_α of ~11° as determined by the EMD method may not be an intrinsic structural characteristic of the native PM but an induced one. The differences in the Δθ_α value due to glucose embedment as determined by the two different approaches (23 vs. 11°) may be attributed to the drastic differences in the experimental conditions used, especially temperature. It is expected that at extremely low temperatures protein dynamics would be highly restricted and Δθ_α relatively curtailed. It is concluded that glucose may not be as benign to biological structures as has been assumed in the past.     

Structural studies on lens proteins

The sequence around the thiol group in lens proteins has been investigated. The proteins were converted into their carboxy[¹⁴C]methyl derivatives and submitted to partial acid hydrolysis, or digested with proteolytic enzymes. Acid hydrolysis of bovine α-crystallin gives N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys (Waley, 1965a), but this dipeptide is not obtained from β-crystallin or γ-crystallin. Trypsin and chymotrypsin also give different peptides from the three crystallins. The radioactive peptide from α-crystallin and chymotrypsin has the sequence Ser-CMCys-Ser-Leu; another peptide, Asp-Leu-Leu-Phe, was also identified. The radioactive peptides obtained from bovine α-crystallin are probably also obtained from human α-crystallin, and from bovine and human albuminoid (the insoluble lens protein). α-Crystallin has been fractionated by chromatography in urea on DEAE-cellulose. Comparison of the fractions by peptide `mapping', and immunochemically, shows that they fall into two classes. The fraction eluted first differs from the later fractions, but the later fractions resemble each other The first fraction may represent impurities, or it may be a structurally different sub-unit of α-crystallin.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F₀ level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λ_max^em ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ_max^em = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ_max^em = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ_max^em = 685 nm) derives from dead chlorophyll. (b) At the F_max level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F₀ level, from closed PS II α-centers (τ ≈ 2.2 ns, λ_max^em = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ_max^em = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).     

Particle Size of Latex Beads Dictates IL-1β Production Mechanism

Macrophages (Mϕ) are well documented to produce IL-1β through various signaling pathways in response to small particles such as silica, asbestos and urea crystals, in the presence of lipopolysaccharide (LPS). However, it has not been clear to what extent particle size affects the response. To investigate this point, we stimulated bone marrow-derived macrophages (BMDM) with size-defined latex beads (LxB). Although both nano-sized (20 nm) and micro-sized (1,000 nm) LxB induced IL-1β production, only the nano-sized particles formed large intracellular vacuoles. In contrast, 100 nm LxB did not induce either of the responses. The same cellular responses were also observed in primary microglia cells. Although K⁺ efflux and NLRP3 activation in BMDM were crucial in response to both 20 and 1,000 nm LxB, only IL-1β production by 20 nm LxB was sensitive to cathepsin B and P2X₇, a receptor for ATP. The response by 1,000 nm LxB relied on a robust production of reactive oxygen species (ROS), since IL-1β production was remarkably reduced by ROS inhibitors such as diphenylene iodonium (DPI) and N-acetylcysteine (NAC). In contrast, IL-1β production by 20 nm LxB was augmented by NAC and in BMDM deficient in thioredoxin-binding protein-2 (TBP-2), a negative regulator of the ROS scavenger thioredoxin. These results suggest that the cells responded differently in their secretion of IL-1β depending on particle size, and that there is a range within which neither pathway works.     

Interactive Effects of Temperature and UV Radiation on Photosynthesis of Chlorella Strains from Polar,Temperate and Tropical Environments: Differential Impacts on Damage and Repair

Global warming and ozone depletion, and the resulting increase of ultraviolet radiation (UVR), have far-reaching impacts on biota, especially affecting the algae that form the basis of the food webs in aquatic ecosystems. The aim of the present study was to investigate the interactive effects of temperature and UVR by comparing the photosynthetic responses of similar taxa of Chlorella from Antarctic (Chlorella UMACC 237), temperate (Chlorella vulgaris UMACC 248) and tropical (Chlorella vulgaris UMACC 001) environments. The cultures were exposed to three different treatments: photosynthetically active radiation (PAR; 400–700 nm), PAR plus ultraviolet-A (320–400 nm) radiation (PAR + UV-A) and PAR plus UV-A and ultraviolet-B (280–320 nm) radiation (PAR + UV-A + UV-B) for one hour in incubators set at different temperatures. The Antarctic Chlorella was exposed to 4, 14 and 20°C. The temperate Chlorella was exposed to 11, 18 and 25°C while the tropical Chlorella was exposed to 24, 28 and 30°C. A pulse-amplitude modulated (PAM) fluorometer was used to assess the photosynthetic response of microalgae. Parameters such as the photoadaptive index (E_k) and light harvesting efficiency (α) were determined from rapid light curves. The damage (k) and repair (r) rates were calculated from the decrease in ΦPSII_eff over time during exposure response curves where cells were exposed to the various combinations of PAR and UVR, and fitting the data to the Kok model. The results showed that UV-A caused much lower inhibition than UV-B in photosynthesis in all Chlorella isolates. The three isolates of Chlorella from different regions showed different trends in their photosynthesis responses under the combined effects of UVR (PAR + UV-A + UV-B) and temperature. In accordance with the noted strain-specific characteristics, we can conclude that the repair (r) mechanisms at higher temperatures were not sufficient to overcome damage caused by UVR in the Antarctic Chlorella strain, suggesting negative effects of global climate change on microalgae inhabiting (circum-) polar regions. For temperate and tropical strains of Chlorella, damage from UVR was independent of temperature but the repair constant increased with increasing temperature, implying an improved ability of these strains to recover from UVR stress under global warming.     

Fusion Peptides Promote Formation of Bilayer Cubic Phases in Lipid Dispersions. An X-Ray Diffraction Study

Small angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the H_II phase and shifted the L_α–H_II transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, Q_II, which becomes inserted between the L_α and H_II phases on the temperature scale. Peptide-induced Q_II had strongly reduced lattice constants in comparison to the Q_II phases that form in pure lipids. Q_II formation was favored at the expense of both L_α and H_II phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the L_α phase and completely abolished the H_II phase in DOPE-Me/WT-20 50:1 dispersions, converted the Q_II phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative Gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving H_II phase-like intermediates.     

Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH₂, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH₂, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state ¹⁹F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by ³¹P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.     

Structure-Function Elucidation of a New α-Conotoxin,Lo1a,from Conus longurionis

α-Conotoxins are peptide toxins found in the venom of marine cone snails and potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors forming ligand-gated ion channels in the plasma membranes of certain neurons and the neuromuscular junction. Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson disease, and Alzheimer disease. To expand the knowledge concerning cone snail toxins, we examined the venom of Conus longurionis. We isolated an 18-amino acid peptide named α-conotoxin Lo1a, which is active on nAChRs. To the best of our knowledge, this is the first characterization of a conotoxin from this species. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs expressed in Xenopus laevis oocytes. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. Lo1a, a member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α₇ nAChRs with an IC₅₀ of 3.24 ± 0.7 μm. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. Because Lo1a has an unusual C terminus, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, to investigate the influence of the C-terminal residue. Lo1a-ΔD has a C-terminal Asp deletion, whereas in Lo1a-RRR, a triple-Arg tail replaces the Asp. They blocked the neuronal nAChR α₇ with a lower IC₅₀ value, but remarkably, both adopted affinity for the muscle subtype α₁β₁δϵ.