Antiparallel Β-structure Research Articles

The Double-Layered Structure of Amyloid-β Assemblage on GM1-Containing Membranes Catalytically Promotes Fibrillization.

Alzheimer's disease (AD) is associated with progressive accumulation of amyloid-β (Aβ) cross-β fibrils in the brain. Aβ species tightly associated with GM1 ganglioside, a glycosphingolipid abundant in neuronal membranes, promote amyloid fibril formation; therefore, they could be attractive clinical targets. However, the active conformational state of Aβ in GM1-containing lipid membranes is still unknown. The present solid-state nuclear magnetic resonance study revealed a nonfibrillar Aβ assemblage characterized by a double-layered antiparallel β-structure specifically formed on GM1 ganglioside clusters. Our data show that this unique assemblage was not transformed into fibrils on GM1-containing membranes but could promote conversion of monomeric Aβ into fibrils, suggesting that a solvent-exposed hydrophobic layer provides a catalytic surface evoking Aβ fibril formation. Our findings offer structural clues for designing drugs targeting catalytically active Aβ conformational species for the development of anti-AD therapeutics.

Effect of nanoparticles on the aggregation and amyloid formation of amyloid-beta peptide.

Nanoparticles are becoming more and more important in medicine and biotechnology. Moreover, due to environmental pollution, the human body is exposed to nanoparticles in the everyday life. Nanomaterials can also affect the aggregation processes of proteins. The Aβ peptides are known for forming amyloid aggregates in the brain in Alzheimer's disease. We used in-house expressed recombinant Aβ(1-42) to study the structure, aggregation kinetics and cytotoxicity of the peptide. We characterized nanoparticles of different materials and studied their effects on the structure and aggregation of Aβ. We followed amyloid formation by monitoring Thioflavin T fluorescence in the presence of nanoparticles. We observed that nanoparticles typically reduce the lag time of aggregation. We found that polystyrene particles bind monomers to a certain saturation point and allow only excess monomers to form fibrils. Calcium fluoride (CaF2) nanoparticles do not bind monomers in a similar fashion, but do influence the secondary structure composition of the resulting amyloid fibrils. We used CD spectroscopy and the BeStSel method to determine the secondary structure composition of the different forms of Aβ. While the main characteristics of Aβ fibrils formed alone is parallel β-sheet, in the presence of CaF2, Aβ forms significant amount of antiparallel β-structure as well. We also conducted cytotoxicity experiments to investigate if nanoparticles alter the toxic effect of Aβ on mouse hippocampal cells. Our preliminary findings suggest that studied particles might reduce cytotoxicity of Aβ if applied in very low concentrations.

Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution

An interplay between monomeric and dimeric forms of human epidermal growth factor (EGF) affecting its interaction with EGF receptor (EGFR) is poorly understood. While EGF dimeric structure was resolved at pH 8.1, the possibility of EGF dimerization under physiological conditions is still unclear. This study aimed to describe the oligomeric state of EGF in a solution at physiological pH value. With centrifugal ultrafiltration followed by blue native gel electrophoresis, we showed that synthetic human EGF in a solution at a concentration of 0.1mg/ml exists mainly in the dimeric form at pH 7.4 and temperature of 37°C, although a small fraction of its monomers was also observed. Based on bioinformatics predictions, we introduced the D46G substitution to examine if EGF C-terminal part is directly involved in the intermolecular interface formation of the observed dimers. We found a reduced ability of the resulting EGF D46G dimers to dissociate at temperatures up to 50°C. The D46G substitution also increased the intermolecular antiparallel β-structure content within the EGF peptide in a solution according to the CD spectra analysis that was confirmed by HATR-FTIR results. Additionally, the energy transfer between Tyr and Trp residues was detected by fluorescence spectroscopy for the EGF D46G mutant, but not for the native EGF. This allowed us to suggest the elongation and rearrangement of the intermolecular β-structure that leads to the observed stabilization of EGF D46G dimers. The results imply EGF dimerization under physiological pH value and temperature and the involvement of EGF C-terminal part in this process.

Anti-Parallel β-Hairpin Structure in Soluble Aβ Oligomers of Aβ40-Dutch and Aβ40-Iowa.

The amyloid-β (Aβ) peptides are associated with two prominent diseases in the brain, Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). Aβ42 is the dominant component of cored parenchymal plaques associated with AD, while Aβ40 is the predominant component of vascular amyloid associated with CAA. There are familial CAA mutations at positions Glu22 and Asp23 that lead to aggressive Aβ aggregation, drive vascular amyloid deposition and result in degradation of vascular membranes. In this study, we compared the transition of the monomeric Aβ40-WT peptide into soluble oligomers and fibrils with the corresponding transitions of the Aβ40-Dutch (E22Q), Aβ40-Iowa (D23N) and Aβ40-Dutch, Iowa (E22Q, D23N) mutants. FTIR measurements show that in a fashion similar to Aβ40-WT, the familial CAA mutants form transient intermediates with anti-parallel β-structure. This structure appears before the formation of cross-β-sheet fibrils as determined by thioflavin T fluorescence and circular dichroism spectroscopy and occurs when AFM images reveal the presence of soluble oligomers and protofibrils. Although the anti-parallel β-hairpin is a common intermediate on the pathway to Aβ fibrils for the four peptides studied, the rate of conversion to cross-β-sheet fibril structure differs for each.

The cytoplasmic tail of influenza A/H1N1 virus hemagglutinin is β-structural

Influenza A/H1N1 virus hemagglutinin (HA) is an integral type I glycoprotein that contains a large glycosylated ectodomain, a transmembrane domain, and a cytoplasmic tail (CT) of 10-14 amino acid residues. There are absolutely no data on the secondary or tertiary structure of the HA CT, which is important for virus pathogenesis. Three highly conserved cysteines are post-translationally modified by the attachment of fatty acid residues that pin the CT to the lipid membrane inside the virion. We applied circular dichroism (CD) and fluorescence spectroscopy analysis to examine four synthetic peptides corresponding to 14-15 C-terminal residues of H1 subtype HA (NH2-WMCSNGSLQCRICI-COOH; NH2-FWMCSNGSLQCRICI-COOH), with free or acetaminomethylated cysteines, in the reduced or non-reduced state, at various pH values and temperatures. The CD analysis detected the formation of a β-structure (30–65% according to the new BeStSel algorithm), in addition to an unstructured random coil, in every peptide in various conditions. It was completely or partially recognized as an antiparallel β-structure that was also confirmed by the multi-bounce Horizontal Attenuated Total Reflectance Fourier Transformed Infrared (HATR-FTIR) spectroscopy analysis. According to the experimental data, as well as 3 D modeling, we assume that the amino acid sequence corresponding to the HA CT may form a short antiparallel β-structure under the lipid membrane within a virion. Communicated by Ramaswamy H. Sarma

A nanofiber assembly directed by the non-classical antiparallel β-structure from 4S-(OH) proline polypeptide.

The antiparallel arrangement of two strands of the non-classical β-structure, formed exclusively via cis-4S-(OH) prolyl polypeptide as established by FRET, propagates into self-assembled nanofibers upon conjugation with C12/C14/C16 hydrocarbon chains.

Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27

Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (β/α)8-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes.

An ALS-mutant TDP-43 neurotoxic peptide adopts an anti-parallel β-structure and induces TDP-43 redistribution.

TDP-43 proteinopathies are clinically and genetically heterogeneous diseases that had been considered distinct from classical amyloid diseases. Here, we provide evidence for the structural similarity between TDP-43 peptides and other amyloid proteins. Atomic force microscopy and electron microscopy examination of peptides spanning a previously defined amyloidogenic fragment revealed a minimal core region that forms amyloid fibrils similar to the TDP-43 fibrils detected in FTLD-TDP brain tissues. An ALS-mutant A315E amyloidogenic TDP-43 peptide is capable of cross-seeding other TDP-43 peptides and an amyloid-β peptide. Sequential Nuclear Overhauser Effects and double-quantum-filtered correlation spectroscopy in nuclear magnetic resonance (NMR) analyses of the A315E-mutant TDP-43 peptide indicate that it adopts an anti-parallel β conformation. When added to cell cultures, the amyloidogenic TDP-43 peptides induce TDP-43 redistribution from the nucleus to the cytoplasm. Neuronal cultures in compartmentalized microfluidic-chambers demonstrate that the TDP-43 peptides can be taken up by axons and induce axonotoxicity and neuronal death, thus recapitulating key neuropathological features of TDP-43 proteinopathies. Importantly, a single amino acid change in the amyloidogenic TDP-43 peptide that disrupts fibril formation also eliminates neurotoxicity, supporting that amyloidogenesis is critical for TDP-43 neurotoxicity.

Improved Secondary Structure Determination and Fold Prediction by Circular Dichroism Spectroscopy

Circular dichroism (CD) spectroscopy is a well known technique for the study of the secondary structure of proteins. Although the presently available algorithms reliably estimate the secondary structure of the α-helical proteins, the prediction of β-sheet structure is much less accurate. In the case of proteins with unique β-structures, such as membrane proteins and amyloid fibrils, the secondary structure prediction results are unacceptable. The problem arises from the diversity of β-structures, which results large spectral differences even between proteins with similar overall β-sheet content. We have developed a new algorithm for the accurate estimation of secondary structure contents for a broader range of protein folds with special interest to β-structure rich proteins including amyloid fibrils. The reference spectrum database was expanded with high quality spectra of globular proteins and amyloid fibrils recorded down to 175 nm using synchrotron radiation CD. Our algorithm takes into account the diverse twist of the β-sheets that has a great influence on the spectral features. For the first time, we can reliably distinguish parallel and antiparallel β-structure using CD spectroscopy. Moreover, it performs better than any other algorithms for any secondary structure types. With its improved accuracy and more detailed structural information, the method is capable of predicting the protein fold to the level of topology in the CATH classification with good reliability. A web server was constructed to make the algorithm available for the protein science research community.

Structure and Dynamics of Cardiotoxins

Cytotoxins (or cardiotoxins; CTs) are toxins from cobra venom characterized by the three-finger (TF) fold. CTs are on average 60-residue-long peptides, possessing as many as 4 disulfide bonds. The elements of antiparallel β-structure take origin from the hydrophobic core formed by the disulfides. The β-strands adopt the shape of the three loops, giving the name of the fold. While neurotoxins (NTs) - also TF proteins from snake venom - exert their effect through specific interactions with protein receptors, no specific protein target has been found for CTs. Unlike NTs, CTs are amphiphilic and cytotoxic against a variety of cells, including cancer ones. Thus, the hypothesis that the activity of CTs is caused by their interactions with lipid membranes is currently central. To understand molecular basis behind variations in toxicities of CTs highly homologous in their sequences, detailed knowledge of their structure and dynamics is required. The present review summarizes experimental and computational data on the spatial organization of CTs and their dynamics in various environments (aqueous solution, membranous milieus).

The intrinsic stability of the human prion β-sheet region investigated by molecular dynamics

Human prion diseases are neurodegenerative disorders associated to the misfolding of the prion protein (PrP). Common features of prion disorders are the fibrillar amyloid deposits and the formation of prefibrillar oligomeric species also suggested as the origin of cytotoxicity associated with diseases. Although the process of PrP misfolding has been extensively investigated, many crucial aspects of this process remain unclear. We have here carried out a molecular dynamics study to evaluate the intrinsic dynamics of PrP β-sheet, a region that is believed to play a crucial role in prion aggregation. Moreover, as this region mediates protein association in dimeric assemblies frequently observed in prion crystallographic investigations, we also analyzed the dynamics of these intermolecular interactions. The extensive sampling of replica exchange shows that the native antiparallel β-structure of the prion is endowed with a remarkable stability. Therefore, upon unfolding, the persistence of a structured β-region may seed molecular association and influence the subsequent phases of the aggregation process. The analysis of the four-stranded β-sheet detected in the dimeric assemblies of PrP shows a tendency of this region to form dynamical structured states. The impact on the β-sheet structure and dynamics of disease associated point mutations has also been evaluated.

The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

Typically as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Phosphorylation stabilizes the ionic and hydrophobic interactions between monomers. Recent studies have shown that some response regulators retain functional activity in the absence of phosphorylation and are termed atypical response regulators. The two currently available receiver domain structures of atypical response regulators are very similar to their phospho-accepting homologs, and their propensity to form homodimers is generally retained. An atypical response regulator, ChxR, from Chlamydia trachomatis, was previously reported to form homodimers; however, the residues critical to this interaction have not been elucidated. We hypothesize that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. To test this hypothesis, structural and functional studies were performed on the receiver domain of ChxR. Two crystal structures of the receiver domain were solved with the recently developed method using triiodo compound I3C. These structures revealed many characteristics unique to OmpR/PhoB subfamily members: typical or atypical. Included was the absence of two α-helices present in all other OmpR/PhoB response regulators. Functional studies on various dimer interface residues demonstrated that ChxR forms relatively stable homodimers through hydrophobic interactions, and disruption of these can be accomplished with the introduction of a charged residue within the dimer interface. A gel shift study with monomeric ChxR supports that dimerization through the receiver domain is critical for interaction with DNA.

К вопросу о выборе потенциальных полей для изучения молекулярной динамики ионных пептидов и их димеров

The paper presents comparative data on the impact of force-fields AMBER (ff03, ff99SB, and ff96) on the results of molecular dynamics experiments with dimeric molecules formed by ion-peptide NH2-(RADA)4-COOH in the β-conformation at two temperatures (300K and 320K). It is shown that an MD simulation in explicit water environment is the most informative approach. The use of different force-fields has a significant influence on the stability of the initial molecular conformation of the peptide over time. Finally, the simulation in ff99SB environment provides significant stability of antiparallel β-structure of the dimer at 300K, while ff96 not only ensures the highest stability of the initial b-peptide conformation at higher temperatures, but also enhances the retention of antiparallel β-conformation, which determines the ability of NH2-(RADA)4-COOH peptides to self-organization.

The Unstructured N-terminal Tail of ParG Modulates Assembly of a Quaternary Nucleoprotein Complex in Transcription Repression

ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the interaction with DNA, and unstructured N-terminal domains of previously unknown function. Here the ParG protein is shown to be a transcriptional repressor of the parFG genes. The protein assembles on its operator site initially as a tetramer (dimer of dimers) and, at elevated protein concentrations, as a pair of tetramers. Progressive deletion of the mobile N-terminal tails concomitantly decreased transcriptional repression by ParG and perturbed the DNA binding kinetics of the protein. The flexible tails are not necessary for ParG dimerization but instead modulate the organization of a higher order nucleoprotein complex that is crucial for proper transcriptional repression. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Numerous ParG homologs encoded by plasmids of Gram-negative bacteria similarly are predicted to possess N-terminal disordered tails, suggesting that this is a common feature of partition operon autoregulation. The results provide new insights into the role of natively unfolded domains in protein function, the molecular mechanisms of transcription regulation, and the control of plasmid segregation.

Isolation, properties and partial amino acid sequence of a new actinoporin from the sea anemone Radianthus macrodactylus

A new cytolytic toxin, actinoporin RTX-S II, was isolated from the sea anemone Radianthus macrodactylus with a high degree of purity by a combination of gel filtration, ion-exchange and reverse-phase chromatography. RTX-S II has molecular mass of 19,280 Da and isoelectric point of 10.0. The hemolytic activity of RTX-S II is inhibited by sphingomyelin. RTX-S II had an LD 50 of 70 mg/kg, and is lacking in phospholipase activity. The amino acid composition of this protein contains a high amount of basic and non-polar amino acids and no cysteine. The N-terminal sequence of RTX-S II was determined. The partial amino acid sequence (141 aa) of RTX-S II was deduced based on the cDNA sequence obtained with two oligonucleotides encoding the N-terminal portion of RTX-S II and the internal conserved cytolysin peptide by PCR. A comparison of the RTX-S II cDNA sequence and the rtx-s II gene obtained with the same PCR primers indicates that they are 100% identical at the nucleotide level. It shows that no introns are present in the corresponding region of the rtx-s II gene. Multiple alignments of RTX-S II with known sequences of actinoporins show that RTX-S II is highly homologous to magnificalysin II from Heteractis magnifica. The predicted secondary structure of RTX-S II is predominantly anti-parallel β-structure, which is in good agreement with experimental data obtained from other sea anemones–actinoporins.

Crystal Structure of α-Galactosidase from Trichoderma reesei and Its Complex with Galactose: Implications for Catalytic Mechanism

The crystal structures of α-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, β- d-galactose, have been determined at 1.54 Å and 2.0 Å resolution, respectively. The α-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the α-galactosidase consists of two domains, an N-terminal catalytic domain of the (β/α) 8 barrel topology and a C-terminal domain which is formed by an antiparallel β-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the α-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the α-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the α-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Crystal Structure of Thermotoga maritima 4-α-Glucanotransferase and its Acarbose Complex: Implications for Substrate Specificity and Catalysis

4-α-Glucanotransferase (GTase) is an essential enzyme in α-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-α- d-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6 Å and 2.5 Å resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (β/α) 8 barrel topology (domain A), a 65 residue domain, domain B, inserted between strand β3 and helix α6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel β-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (−2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (β/α) 8 barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic “clamp” that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from −2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from −2 to +2, play the dominant role in enzyme–substrate recognition, consistent with the observation that the smallest donor for T. maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using 13C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41 000 (FP; residues 1–23; AVGIGALFLGFLGAAGSTMGARSCONH 2) participates in fusion processes underlying virus–cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy indicated the following α-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes∼hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). 12C-FTIR spectra also showed disordered FP structures in these environments, along with substantial β-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with 13C-carbonyl at multiple sites. Combining these 13C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: α-helix (residues 3–16) and random and β-structures (residues 1–2 and residues 17–23). Additional 13C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the α-helix extends over residues 1–16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, 13C-FTIR spectroscopy showed α-helical conformations for the central core of FP (residues 5–15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel β-structure. FP at low loading in ghosts probably inserts deeply as an α-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, β-sheet. In future studies, 13C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.

Destabilization and Fusion of Zwitterionic Large Unilamellar Lipid Vesicles Induced by a β-Type Structure of the Hiv-1 Fusion Peptide

The peptide HIVarg, corresponding to a sequence of 23 amino acid residues at the N-terminus of HIV-1 gp41, has the capacity to induce fusion of large unilamellar vesicles (LUV) consisting of negatively charged or zwitter-ionic phospholipids. In the present study, we further characterize this destabilization and fusion process using LUV consisting of phosphatidylcholine, phosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Evidence for fusion includes a demonstration of membrane lipid mixing as well as mixing of aqueous vesicle contents. Kinetic analysis of the overall process of vesicle aggregation and fusion revealed that the rate constant of the fusion step per se increased dramatically with the peptide-to-lipid molar ratio, indicating that the peptide acts as a true fusogen. The peptide caused the release of small molecules (Ants/DPX), whereas large solutes (Fitc-dextran, MWav 19,600) were partly retained. The estimated critical number of peptides per vesicle necessary to release vesicle contents, M = 2-4, indicates that leakage does not involve the formation of classical pores. Infrared spectroscopy of the peptide in the presence of liposomes demonstrated that the equilibrium conformation of the membrane-bound peptide is an antiparallel β-structure. This finding supports the notion that the HTV fusion peptide in a β-conformation has the capacity to perturb vesicle bilayers, inducing initial permeabilization and subsequent membrane fusion.

Differential Regulation of GTPase Activity by Mastoparan and Galparan

The chimeric peptide galparan, composed of galanin(1-13) in the N-terminus and mastoparan in the C-terminus, was recently designed and synthesized. The effect of galparan on GTPase activity of rat brain cortical membranes was studied in comparison with the effect of mastoparan and galanin. GTPase was activated by mastoparan but it was noncompetitively inhibited by galparan, while no effect of galanin and galanin(1-13) was found in this tissue. EC50of 12.1 ± 2.1 μM and Hill coefficient of 2.1 ± 0.6 was calculated for galparan from a dose–response curve andKiof 19.1 ± 0.3 μM was obtained by fitting the experimental data to the Michaelis–Menten equation valid in the presence of noncompetitive inhibitor. Mastoparan reversed the effect of galparan in a fully competitive manner while benzalkonium chloride did not prevent the inhibition of GTPase activity by galparan. Pertussis-toxin-catalyzed ribosylation of G proteins from rat brain cortical membranes resulted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose–response curve for galparan inhibition. The rate of GTPγS binding to G proteins from rat brain cortical membranes was not influenced by galparan. CD spectra revealed predominantly antiparallel β-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid vesicles it adopted a higher amount of α-helix. Critical micelle concentration of galparan in buffer solution of 22 μM was determined. It is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these two peptides on G proteins.