Functional domains of a pore-forming cardiotoxic protein, volvatoxin A2

Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the mushroom Volvariella volvacea. We identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 as a domain for oligomerization by limited tryptic digestion. On preincubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site-directed mutagenesis, the amphipathic alpha-helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the recombinant C-terminal fragment (128-199 residues) was found to be a functional domain that mediates the membrane binding of VVA2. We found a fragment localized at the C-terminal half of VVA2 containing beta6, -7, and -8, which is protected from protease digestion because of its insertion of a membrane. We also identified a putative heparin binding site (HBS) located in the VVA2 C terminus (166-194 residues), which was conserved among 10 kinds of snake venom cardiotoxins. VVA2 or the reHBS fragment was shown to interact with sulfated glycoaminoglycans by affinity column chromatography. The finding of a higher number of glycoaminoglycans in the membrane of cardiac myocytes suggested that they could be the specific membrane target for VVA2. Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion.     

Activation of crotoxin B by volvatoxin A2

Of the two proteins making up volvatoxin, a mushroom toxin, the smaller, volvatoxin A2, of molecular weight 25,000, is able to synergistically increase the low neurotoxicity of crotoxin B, the larger basic component of the $\text{Crotalus}\text{durissus}\text{terrificus}$ neurotoxin, crotoxin. No other combination of volvatoxin and crotoxin components shows this effect. Gel filtration suggests that the stable complex is composed of one molecule of each, crotoxin B and volvatoxin A2, although highest activation may be favored by the presence of an additional molecule of crotoxin B. Upon sucrose density gradient electrophoresis, the complex of volvatoxin A2 and crotoxin B behaves as a single species of greater anodic mobility than volvatoxin A2 alone. The complex formation between these two toxin components is not a nonspecific interaction of an acidic with a basic protein, since crotoxin B is not activated, nor complexed by other proteins of similar isoelectric points to volvatoxin A2.     

Crystallization and preliminary X-ray analysis of volvatoxin A2 from Volvariella volvacea

Volvatoxin A2, an ion channel disturbed cardiotoxic and hemolytic protein from the edible mushroom, Volvarilla volvacea, has been crystallized by the vapor diffusion method using polyethylene glycol 4000 and ammonium sulfate in sodium acetate buffer pH 4.6. The best crystals belong to the monoclinic space group C2 with unit cell dimensions a = 155.25 Å, b = 58.06 Å, c = 116.92 Å, and β = 119.5°. These crystals diffract to at least 2.2 Å and there are four molecules of molecular weight 24 kDa per asymmetric unit with a solvent content of 48%.     

Crystal structures and electron micrographs of fungal volvatoxin A2

Membrane adhesion and insertion of protein are essential to all organisms, but the underlying mechanisms remain largely unknown. Membrane pore-forming toxins (PFTs) are potential model systems for studying these mechanisms. We have determined the crystal structures of volvatoxin A2 (VVA2), a fungal PFT from Volvariella volvacea, using Br-multiple-wavelength anomalous diffraction (MAD). The VVA2 structures obtained at pH 4.6, pH 5.5 and pH 6.5 were refined to resolutions of 1.42 A, 2.6 A and 3.2 A, respectively. The structures reveal that the VVA2 monomer contains a single alpha/beta domain. Most of the VVA2 surface is occupied by its oligomerization motif and two putative heparin-binding motifs. Residues Ala91 to Ala101 display several conformations at different pH values, which might be under the control of His87. We also found that the shape of one putative heparin-binding motif in VVA2 appears similar to those found in fibroblast growth factors, and the other one displays a linear polypeptide. Our results suggest several possible intermediates of protein assembly in solution and protein adhering to cell membranes before conformational changes. The electron micrographs of VVA2 molecules in solution, at a protein concentration of 1 microg ml(-1), show that they can assemble into filament-like or braid-like oligomers in a pH-dependent way. In addition, the arc-shaped VVA2 structure obtained at pH 6.5 suggests that VVA2 could form a two-layered helical oligomer with 18 subunits per turn. The structures presented here could be used to elucidate the pore-formation mechanisms of VVA2 and its structural neighbors, Cyt toxins from Bacillus thuringiensis.     

Amphipathic alpha-helices in proteins: results from analysis of protein structures

Amphipathic alpha-helices play a crucial role in mediating the interaction of peptides and proteins with membranes. We have analyzed protein structures for the occurrence of 18-residue amphipathic helices. We find several of these alpha-helices having average hydrophobic moments and average hydrophobicities that would favor their interaction with membranes. We have analyzed the distribution of net charge, helix length, normalized frequency of occurrence, and propensities of the 20 amino acids in the delineated 18-residue helices. We have observed distinct differences in the frequencies of occurrence of polar and hydrophobic amino acids at positions 1-18 in amphipathic and nonamphipathic helices. There are also differences in propensities of the 20 amino acids to occur at positions 1-18 of amphipathic and nonamphipathic helices. Synthetic peptides corresponding to some of these surface-seeking helices do possess antibacterial and/or hemolytic activities. Knowledge of the distribution of charges in 18-residue surface-seeking amphipathic alpha-helices, as well as propensity of occurrence of amino acids at various positions, would be useful inputs in the de novo design of amphipathic peptides.     

Strong conformational propensities enhance T cell antigenicity

The ability to predict T cell antigenic peptides would have important implications for the development of artificial vaccines. As a first step towards prediction, this report uses a new statistical technique to discover and evaluate peptide properties correlating with T cell antigenicity. This technique employs Monte Carlo computer experiments and is applicable to many problems involving protein or DNA. The technique is used to evaluate the contribution of various peptide properties to helper T cell antigenicity. The properties investigated include amphipathicities (alpha and beta), conformational propensities (alpha, beta, turn and coil), and the correlates of alpha-helices, such as the absence of helix-breakers and the positioning of the residues which stabilize alpha-helical dipoles. We also investigate segmental amphipathicity. (A peptide has this property when it contains at least two disjoint subpeptides, one hydrophobic, one hydrophilic.) Statistical correlations and stratifications assessed independent contributions to T cell antigenicity. The findings presented here have important implications for the manufacture of peptide vaccines. These implications are as follows: if possible, peptide vaccines should probably be those protein segments which have a propensity to form amphipathic alpha-helices, which do not have regions with a propensity to coil conformations, and which have a lysine at their COOH-terminus. The last two observations are of particular use in manufacturing peptides vaccines: they indicate where the synthetic peptides should be terminated. These implications are supported by the findings given below. The significances (p values) support the following statistical generalites about antigenic conformations: most helper T cell antigenic sites are amphipathic alpha-helices; alpha-helical amphipathicity and propensity to an alpha-helical conformation contribute independently to T cell antigenicity; there is evidence that some T cell antigenic sites are beta conformations instead of alpha-helices; T cell antigenic sites avoid random coiled conformations; and T cell antigenic sites are usually not segmentally amphipathic. alpha-Helical amphipathicity was significant, but segmental amphipathicity was not. This has implications for the dimensions of the structure interacting with the hydrophobic portion of an amphipathic T cell antigenic site. Lysines are unusually frequent at the COOH-terminal of T cell antigenic sites, even after accounting for tryptic digests. These lysines can stabilize alpha-helical peptides by a favorable interaction with alpha-helical dipoles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural determinants that target the hepatitis C virus core protein to lipid droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.     

alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes

alpha-Synuclein, a pathological component of Parkinson's disease by constituting the Lewy bodies, has been suggested to be involved in membrane biogenesis via induction of amphipathic alpha-helices. Since the amphipathic alpha-helix is also known as a recognition signal of calmodulin for its target proteins, molecular interaction between alpha-synuclein and calmodulin has been investigated. By employing a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, alpha-synuclein has been shown to yield a heterodimeric 1 : 1 complex with calmodulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and even absence of calcium, whereas beta-synuclein was more dependent upon calcium for its calmodulin interaction. The selective calmodulin interaction of alpha-synuclein in the absence of calcium was also demonstrated with the aggregation kinetics of the synucleins in which only the alpha-synuclein aggregation was affected by calmodulin. A reversible binding assay confirmed that alpha-synuclein interacted with the Ca2+-free as well as the Ca2+-bound calmodulins with almost identical Kds of 0.35 micro m and 0.31 micro m, respectively, while beta-synuclein preferentially recognized the Ca2+-bound form with a Kd of 0.68 micro m. By using a C-terminally truncated alpha-synuclein of alpha-syn97, the calmodulin binding site(s) on alpha-synuclein was(were) shown to be located on the N-terminal region where the amphipathic alpha-helices have been suggested to be induced upon membrane interaction. By employing liposome and calmodulin in a state of being either soluble or immobilized on agarose, actual competition of alpha-synuclein between membranes and calmodulin was demonstrated with the observation that alpha-synuclein previously bound to the liposome was released upon specific interaction with the calmodulins. Taken together, these data may suggest that alpha-synuclein could act not only as a negative regulator for calmodulin in the presence and even absence of calcium, but it could also exert its activity at the interface between calmodulin and membranes.     

Nitration and chlorination of folic acid by peroxynitrite and hypochlorous acid,and the selective binding of 10-nitro-folate to folate receptor beta

Peptides pore-forming in cell membrane have been identified from a wide range of animals. A putative pore-forming peptide deduced from a cDNA clone of Clonorchis sinensis (clonorin) was predicted to consist of four amphipathic alpha-helices. Clonorin contained six invariably conserved cysteine residues, identified to form three disulfide bonds. These predicted structural features are highly homologous with pore-forming peptides, the amoebapores. Recombinant clonorin showed hemolytic activity toward rabbit erythrocytes. The hemolytic activity of C. sinensis extract increased dose-dependently and was inhibited by anti-clonorin immune sera. The clonorin was expressed developmentally in juvenile and adult flukes and localized in the intestinal epithelium of adult flukes. It is proposed that, through lysing host cellular components, clonorin could enhance proteolytic digestion in the intestine of C. sinensis.     

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Expression and functional characterization of bluetongue virus VP5 protein: role in cellular permeabilization

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione S-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis.     

Membranotropic effect of some triterpene glycosides possessing immunostimulating properties

The peculiarities of the interaction between cell membrane lipids and triterpene glycosides from holothurians Apostichopus japonicus S. and Cucumaria japonica (holotoxin A1 and cucumarioside A2-2, respectively) were studied in comparison with plant saponins from Quillaja saponaria, known as hemolytic, adjuvant, and structure-forming components of immunostimulating complexes. Similar to Quillaja saponins, the sea glycosides, holotoxin A1 and cucumarioside A2-2 were shown to possess a high hemolytic activity (2.6 and 3 microg/ml, respectively) and sterol-depending membranotropic effect mediated by the formation of nonbilayer sterol-lipid-glycoside complexes. At the same time, cucumarioside A2-2 bound exogenic cholesterol only in the presence of membrane lipids, such as phosphatidylcholine or monogalactosyldiacylglycerol, in contrast to Quillaja saponins and holotoxin A1, which bound cholesterol in the molar ratios 1:2 and 1:8, respectively. Moreover, in all cases, tree-component complexes containing cholesterol, lipid, and glycoside exhibited a lower hemolytic activity compared with two-component sterol-glycoside complexes. It was concluded that the hydrophobic medium of cell membranes performs a potentiative role in the effective interaction between triterpene glycosides and "sterol receptors". A method for decreasing the toxicity of membranotropic holothurian glycosides possessing the immunomodulating properties was suggested.     

Apolipoprotein E: phospholipid binding studies with synthetic peptides from the carboxyl terminus.

We have previously shown that the synthetic peptide apoE(129-169) forms lipid-peptide complexes with dimyristoylphosphatidylcholine (DMPC) with an L:P molar ratio of 125:1; the peptide in the isolated complex contains approximately 56% alpha-helicity. These results verify the presence of an amphipathic alpha-helix in this region of apoE as predicted by Chou-Fasman analysis and hydrophobicity calculations. To further define the lipid binding regions of apoE, we have synthesized four peptides, apoE(211-243), -(202-243), -(267-286), and -(263-286), from the carboxyl terminus of apoE and studied their lipid binding properties; apoE(202-243) contains two potential amphipathic helices. Although all four peptides formed alpha-helices in the helix-forming solvent 30% hexafluoropropanol, we found that only apoE(263-286) formed a stable complex with DMPC. The peptide contained approximately 80% alpha-helicity, and its Trp fluorescence spectrum was blue-shifted by 20 nm in the complex which had an L:P ratio of 163:1. We conclude that this sequence is a newly identified lipid binding region of apoE and that the amphipathic helices 203-221 and 226-243 are too hydrophilic to bind phospholipid.     

The effect of cyclization of magainin 2 and melittin analogues on structure, function, and model membrane interactions: implication to their mode of action

The amphipathic alpha-helical structure is a common motif found in membrane binding polypeptides including cell lytic peptides, antimicrobial peptides, hormones, and signal sequences. Numerous studies have been undertaken to understand the driving forces for partitioning of amphipathic alpha-helical peptides into membranes, many of them based on the antimicrobial peptide magainin 2 and the non-cell-selective cytolytic peptide melittin, as paradigms. These studies emphasized the role of linearity in their mode of action. Here we synthesized and compared the structure, biological function, and interaction with model membranes of linear and cyclic analogues of these peptides. Cyclization altered the binding of melittin and magainin analogues to phospholipid membranes. However, at similar bound peptide:lipid molar ratios, both linear and cyclic analogues preserved their high potency to permeate membranes. Furthermore, the cyclic analogues preserved approximately 75% of the helical structure of the linear peptides when bound to membranes. Biological activity studies revealed that the cyclic melittin analogue had increased antibacterial activity but decreased hemolytic activity, whereas the cyclic magainin 2 analogue had a marked decrease in both antibacterial and hemolytic activities. The results indicate that the linearity of the peptides is not essential for the disruption of the target phospholipid membrane, but rather provides the means to reach it. In addition, interfering with the coil-helix transition by cyclization, while maintaining the same sequence of hydrophobic and positively charged amino acids, allows a separated evaluation of the hydrophobic and electrostatic contributions to binding of peptides to membranes.     

A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.     

A carboxy-terminal fragment of protein mu 1/mu 1C is present in infectious subvirion particles of mammalian reoviruses and is proposed to have a role in penetration.

Penetration of a cell membrane as an early event in infection of cells by mammalian reoviruses appears to require a particular type of viral particle, the infectious subvirion particle (ISVP), which is generated from an intact virion by proteolytic cleavage of the outer capsid proteins sigma 3 and mu 1/mu 1C. Characterizations of the structural components and properties of ISVPs are thus relevant to attempts to understand the mechanism of penetration by reoviruses. In this study, a novel, approximately 13-kDa carboxy-terminal fragment (given the name phi) was found to be generated from protein mu 1/mu 1C during in vitro treatments of virions with trypsin or chymotrypsin to yield ISVPs. With trypsin treatment, both the carboxy-terminal fragment phi and the amino-terminal fragment mu 1 delta/delta were shown to be generated and to remain attached to ISVPs in stoichiometric quantities. Sites of protease cleavage were identified in the deduced amino acid sequence of mu 1 by determining the amino-terminal sequences of phi proteins: trypsin cleaves between arginine 584 and isoleucine 585, and chymotrypsin cleaves between tyrosine 581 and glycine 582. Findings in this study indicate that sequences in the phi portion of mu 1/mu 1C may participate in the unique functions attributed to ISVPs. Notably, the delta-phi cleavage junction was predicted to be flanked by a pair of long amphipathic alpha-helices. These amphipathic alpha-helices, together with the myristoyl group at the extreme amino terminus of mu 1/mu 1N, are proposed to interact directly with the lipid bilayer of a cell membrane during penetration by mammalian reoviruses.     

Insect lipoprotein biogenesis depends on an amphipathic beta cluster in apolipophorin II/I and is stimulated by microsomal triglyceride transfer protein

Lipoproteins transport lipids in the circulation of an evolutionally wide diversity of animals. The pathway for lipoprotein biogenesis has been revealed to a large extent in mammals only, in which apolipoprotein B (apoB) acquires lipids via the assistance of microsomal triglyceride transfer protein (MTP) and binds them by means of amphipathic protein structures. To investigate whether this is a common mechanism for lipoprotein biogenesis in animals, we studied the structural elements involved in the assembly of the insect lipoprotein, lipophorin. LOCATE sequence analysis predicted that the insect lipoprotein precursor, apolipophorin II/I (apoLp-II/I), contains clusters of amphipathic alpha-helices and beta-strands, organized along the protein as N-alpha(1)-beta-alpha(2)-C, reminiscent of a truncated form of apoB. Recombinant expression of a series of C-terminal truncation variants of Locusta migratoria apoLp-II/I in an insect cell (Sf9) expression system revealed that the formation of a buoyant high density lipoprotein requires the amphipathic beta cluster. Coexpression of apoLp-II/I with the MTP homolog of Drosophila melanogaster affected insect lipoprotein biogenesis quantitatively as well as qualitatively, as the secretion of apoLp-II/I proteins was increased several-fold and the buoyant density of the secreted lipoprotein decreased concomitantly, indicative of augmented lipidation. Based on these findings, we propose that, despite specific modifications, the assembly of lipoproteins involves MTP as well as amphipathic structures in the apolipoprotein carrier, both in mammals and insects. Thus, lipoprotein biogenesis in animals appears to rely on structural elements that are of early metazoan origin.     

Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution

Colicin B (55 kDa) is a cytotoxic protein that recognizes the outer membrane transporter, FepA, as a receptor and, after gaining access to the cytoplasmic membranes of sensitive Escherichia coli cells, forms a pore that depletes the electrochemical potential of the membrane and ultimately results in cell death. To begin to understand the series of dynamic conformational changes that must occur as colicin B translocates from outer membrane to cytoplasmic membrane, we report here the crystal structure of colicin B at 2.5 A resolution. The crystal belongs to the space group C2221 with unit cell dimensions a = 132.162 A, b = 138.167 A, c = 106.16 A. The overall structure of colicin B is dumbbell shaped. Unlike colicin Ia, the only other TonB-dependent colicin crystallized to date, colicin B does not have clearly structurally delineated receptor-binding and translocation domains. Instead, the unique N-terminal lobe of the dumbbell contains both domains and consists of a large (290 residues), mostly beta-stranded structure with two short alpha-helices. This is followed by a single long ( approximately 74 A) helix that connects the N-terminal domain to the C-terminal pore-forming domain, which is composed of 10 alpha-helices arranged in a bundle-type structure, similar to the pore-forming domains of other colicins. The TonB box sequence at the N-terminus folds back to interact with the N-terminal lobe of the dumbbell and leaves the flanking sequences highly disordered. Comparison of sequences among many colicins has allowed the identification of a putative receptor-binding domain.     

Influence of alpha-helices on the emulsifying properties of proteins

A peptide derived from apomyoglobin by cyanogen bromide cleavage was found to be an active emulsifier. This molecule, peptide 1-55, has two potential amphipathic alpha-helices and a hydrophilic C-terminal domain. The importance of each of these domains to the emulsifying properties of this molecule was investigated by testing the products of gene constructs based on the sequence of peptide 1-55, but lacking one of the three domains. The emulsifying activity of the peptides lacking either of the alpha-helices was correlated with the hydrophobic moments of their respective helices. The hydrophobic moment is a measure of the amphipathicity of alpha-helices; a hydrophobic moment analysis of other emulsifying peptides supports the hypothesis that a high hydrophobic moment contributes to good emulsifying properties in a molecule which contains alpha-helices.