Amphiphilic Α-helices Research Articles

Influence of C-terminal amidation on the antimicrobial and hemolytic activities of cationic α-helical peptides

Abstract The effect of C-terminal amidation on the antimicrobial and hemolytic activities of antimicrobial peptides was studied using three cationic peptides which form amphiphilic α-helices when bound to membranes. The natural antimicrobial peptide PGLa, the designer-made antibiotic MSI-103, and the cell-penetrating "model amphipathic peptide" (MAP) are all amidated in their original forms, and their biological activities were compared with the same sequences carrying a free C-terminus. It was found that, in general, a free COOH-terminus reduces both the antimicrobial activity and the hemolytic side effects of the peptides. The only exception was observed for MSI-103, whose antimicrobial activity was not decreased in the acid form. Having shown that the therapeutic index (TI) of this novel peptide is significantly higher than for the other tested peptides, with high antibiotic activity and little undesired effects, we suggest that it could be a useful starting point for further development of new peptide antibiotics.

Biological activities of synthetic grammistins and analogous peptides

Grammistins, peptide toxins isolated from the skin secretions of two species of soapfishes Grammistes sexlineatus and Pogonoperca punctata, are new members of the family of linear membrane-lytic antibacterial peptides characterized by the abundance of amphiphilic α-helices. As the first step toward future use of grammistins to examine the mode of membrane lysis and design new antibiotics, C-terminally free and amidated forms were chemically synthesized for each of five grammistins (Gs 1, Gs 2, Pp 1, Pp 2b and Pp 3), and evaluated for hemolytic and antibacterial activities. No difference in biological activities was observed between C-terminally free and amidated forms of Gs 1, while the C-terminally amidated forms of the other grammistins exhibited much higher activities than the C-terminally free forms. Comparison with the biological activities of natural grammistins, together with our preliminary cloning experiments, showed that natural grammistins, except for Gs 1, are C-terminally amidated. As for Pp 1, six analogous peptides were further synthesized. Determination of their biological activities revealed that the amphiphilic α-helical structure and positive charge are important for the hemolytic and antibacterial activities, respectively, of Pp 1.

Effects of Synthetic Amphiphilic α-Helical Peptides on the Electrochemical and Structural Properties of Supported Hybrid Bilayers on Gold

Amphiphilic alpha-helices were formed from designed synthetic peptides comprising alanine, phenylalanine, and lysine residues. The insertion of the alpha-helical peptides into hybrid bilayers assembled on gold was studied by a variety of methods to assess the resulting structural characteristics, such as electrical resistance and molecular orientation. Self-assembled monolayers (SAMs) of dodecanethiol (DDT); octadecanethiol (ODT); and 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) were formed on gold substrates with and without incorporated peptide. Supported hybrid bilayers and multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were formed on SAMs by the "paint-freeze" method of bilayer formation. Modeling of electrochemical impedance spectroscopy data using equivalent electrochemical circuits revealed that the addition of peptide decreased dramatically the resistive element of the bilayer films while maintaining the value of the capacitive element, indicating successful incorporation of peptide into a well-formed bilayer. Near-edge X-ray absorption fine structure spectroscopy data provided evidence that the molecules in the SAMs and hybrid multilayers were ordered even in the presence of peptide. The peptide insertion into the SAM was confirmed by observing the pi* resonance peak correlating with phenylalanine and a peak in the nitrogen K-edge regime attributable to the peptide bond.

Chaperomics: In Vivo GroEL Function Defined

A recent proteome analysis of protein folding inside cells of Escherichia coli predicts that only 84 of the approximately 2400 cytosolic proteins expressed in minimal media depend absolutely on the GroEL/GroES chaperone system to avoid aggregation. These proteins are enriched in alpha/beta domains and 13 are essential for growth.

Amphiphilic β-sheet cobra cardiotoxin targets mitochondria and disrupts its network

Recent advance in understanding the role of toxin proteins in controlling cell death has revealed that pro-apoptotic viral proteins targeting mitochondria contain amphiphilic α-helices with pore-forming properties. Herein, we describe that the pore-forming amphiphilic β-sheet cardiotoxins (or cytotoxins, CTXs) from Taiwan cobra ( Naja atra) also target mitochondrial membrane after internalization and act synergistically with CTX-induced cytosolic calcium increase to disrupt mitochondria network. It is suggested that CTX-induced fragmentation of mitochondria play a role in controlling CTX-induced necrosis of myocytes and cause severe tissue necrosis in the victims.

Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic α-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted α-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.

Interaction of grammistins with lipids and their antibacterial activity

: Grammistins are hemolytic and ichthyotoxic peptides in the skin secretion of soapfishes and are structurally characterized by their abundance in amphiphilic α-helicity. In the present study, their interaction with lipids and lipid vesicles as well as antibacterial activity were examined using four grammistins (Gs 1 and Gs 2 from Grammistes sexlineatus and Pp 1 and Pp 3 from Pogonoperca punctata). The hemolytic activity of grammistins was inhibited by phospholipids but not by cholesterol. Moreover, grammistins released carboxyfluorescein entrapped within liposomes made of phosphatidylcholine. In contrast, grammistins were found to have antibacterial activity with a broad spectrum against nine species of bacteria, including both Gram-negative and Gram-positive groups. The potency of their antibacterial activity was not related to that of hemolytic activity, suggesting that grammistins bind to membrane phospholipids but lyse erythrocyte and bacterial membranes via different mechanisms. Conclusively, grammistins are new members of the family of cell non-selective membrane-lytic peptides with amphiphilic α-helices, being similar to pardaxins, which are secreted from the skin of soles, and to melittin, which is derived from bee venom.

Isolation and Characterization of a Novel Protein Toxin from the Hawaiian Box Jellyfish (Sea Wasp) Carybdea alata

The box jellyfish (sea wasp) Carybdea alata Reynaud, 1830 (Cubozoa) is distributed widely in the tropics. The sting of C. alata causes severe pain and cutaneous inflammation in humans. We successfully isolated C. alata toxin-A (CaTX-A, 43 kDa) and -B (CaTX-B, 45 kDa) for the first time from the tentacle of C. alata collected at a site along the Hawaiian shore. The experimental results showed that CaTX-A, but not CaTX-B, is present in the nematocyst, the organ responsible for stinging. Both CaTX-A and -B showed potent hemolytic activity, with CaTX-A being lethally toxic to crayfishes when administered via intraperitoneal injection. Furthermore, we sequenced the cDNA encoding CaTX-A. The deduced amino acid sequence of CaTX-A (463 amino acids) showed 43.7% homology to Carybdea rastoni toxins (CrTXs) but not with any other known proteins. Therefore, these jellyfish toxins potentially represent a novel class of bioactive proteins. Secondary structure analysis of CaTX-A and CrTXs suggested the presence of amphiphilic α-helices, which are also seen in several known hemolytic or cytolytic protein toxins, including peptide toxins.

Isolation and structures of grammistins, peptide toxins from the skin secretion of the soapfish Grammistes sexlineatus

Two peptide toxins (named grammistins Gs 1 and Gs 2) with hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Grammistes sexlineatus. Grammistin Gs 2 showed 6–11× higher hemolytic activity and 10× higher ichthyotoxicity than grammistin Gs 1. The complete amino acid sequences of Gs 1 comprising 25 residues and Gs 2 comprising 24 residues were determined. Although a search by the database failed to find any homologous toxins from other sources, the grammistins were similar in secondary structures as well as biological activities to the two classes of peptide toxins, melittin from the bee venom and pardaxins from the skin secretion of two species of soles. CD experiments and helical wheel projections showed that the grammistins were randomly coiled in distilled water but formed amphiphilic α-helices in the presence of SDS micelles. In addition, they were found to be surface seeking peptides by the Eisenberg plot and assumed to exist as aggregates of 3–4 molecules. Interestingly, grammistin Gs 2 is much more abundant in amphiphilic α-helices and much higher in biological activities than melittin and pardaxins as well as grammistin Gs 1.

Phase Transitions in Monolayers of Human Apolipoprotein C-I

Human apolipoprotein C-I was deposited onto a highly ionic water subphase to form a monolayer in a Langmuir trough. Pressure−area isotherms of the human apolipoprotein C-I monolayer, as well as direct observations with Brewster angle microscopy, were carried out. Two first-order phase transitions were found. The first phase transition involves the coexistence of a fluid condensed phase and a gas phase, whereas the second one is a transition between two condensed phases. A protein model is proposed, where the apolipoprotein C-I is made of two amphiphilic α-helices bonded with a poorly structured polypeptide fragment. With this model, we have been able to explain our results, in particular the second phase transition, which seems to correspond to a conformational change in the protein. This conformational change could be due to the desorption of one amphiphilic α-helix from the subphase, when lateral pressure is applied to the protein monolayer. The relevance of this conformational change for lipid binding activity is discussed.

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

An investigation into the lipid interactions of peptides corresponding to the C-terminal anchoring domains of Escherichia coli penicillin-binding proteins 4, 5 and 6

The Escherichia coli low molecular mass penicillin-binding proteins PBP4, PBP5 and PBP6 are DD-peptidases involved in murein biosynthesis. It has been suggested that these proteins may be anchored to the periplasmic face of the inner membrane via their C termini. Here, peptide homologues (P4, P5 and P6) of the PBP4, PBP5 and PBP5 C-terminal regions have been used to investigate potential protein-lipid interactions involved in this anchoring mechanism. Surface pressure changes observed for the interactions of P5 and P6 with a range of monolayers indicated that the peptides are membrane interactive and that the interactions proceeded via predominantly hydrophobic forces with only minor requirements for anionic lipid. In contrast, P4 interactions with monolayers appeared to proceed via predominantly electrostatic forces with a major requirement for anionic lipid. The lipid interactions of all three peptides were generally enhanced by low pH and for P5 and P6 were in the range of 10–15 mN m −1 whereas for P4 interactions they were in the range of 3–7 mN m −1. CD analysis implied the presence of α-helical structure in P5 and P6 and molecular area determinations implied that P4 may also possess helical architecture in the presence of dioleoylphosphatidylglycerol monolayers. Overall, our results support the view that C-terminal amphiphilic α-helices are involved in the membrane anchoring of PBP5 and PBP6 and suggest that a similar mechanism could contribute to PBP4-membrane anchoring. Furthermore, we have speculated that the presence of cationic residues in the hydrophilic face of these α-helices may help facilitate membrane interaction.

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR-G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125-137), the N-terminal region of the third intracellular loop (217-227) and the juxtamembranous region of the C-terminal tail (304-316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217-227 and 304-316) are predicted to form amphiphilic alpha-helices. Only the peptide representing residues 217-227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

α-Helical conformation in the C-terminal anchoring domains of E. coli penicillin-binding proteins 4, 5 and 6

The E. coli low molecular mass penicillin-binding proteins (PBP's) are penicillin sensitive, enzymes involved in the terminal stages of peptidoglycan biosynthesesis. These PBP's are believed to anchor to the periplasmic face of the inner membrane via C-terminal amphiphilic α-helices but to date the only support for this hypothesis has been obtained from theoretical analysis. In this paper, the conformational behaviour of synthetic peptides corresponding to these C-terminal anchoring domains was studied as a function of solvent, pH, sodium dodecyl sulphate micelles and phospholipid (DOPC, DOPG) vesicles using circular dichroism (CD) spectroscopy. The CD data showed that in 2,2,2-trifluoroethanol or sodium dodecylsulphate, all three peptides have the capacity to form an α-helical conformation but in aqueous solution or in the presence of phospholipid vesicles only those peptides corresponding to the PBP5 and PBP6 C-termini were observed to do so. A pH dependent loss of α-helical conformation in the peptide corresponding to the PBP5 C-terminus was found to correlate with the susceptibility of PBP5 to membrane extraction. This correlation would agree with the hypothesis that an α-helical conformation is required for membrane interaction of the PBP5 C-terminal region.

An investigation into the ability of C-terminal homologues of Escherichia coli low molecular mass penicillin-binding proteins 4, 5 and 6 to undergo membrane interaction

The Escherichia coli low molecular mass penicillin-binding proteins (PBP4, PBP5 and PBP6) are a group of penicillin-sensitive enzymes involved in the final stages of cell wall assembly. It has been suggested that these proteins may interact with the periplasmic face of the inner membrane via C-terminal amphiphilic α-helices. Theoretical analysis has predicted that these C-terminal helical regions may be membrane interactive. We have tested this hypothesis by assaying PBP C-terminal homologues (P4, P5 and P6) for haemolytic activity. Our results show that the PBP5 and PBP6 C-terminal homologues readily lyse sheep erythrocytes in a pH-dependent manner with LD 50's of 3.5 × 10 −6 M and 6.8 × 10 −7 M respectively at pH 7. These results appear to support the present model for the membrane anchoring of PBP5 and PBP6. The PBP4 C-terminal homologue shows no evidence of haemolytic activity which could imply a different means of membrane association for PBP4.

Structure of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 A resolution: an enzyme catalyst generated via adaptive mutation.

Here we describe the three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3. This enzyme catalyzes the hydrolysis of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA. The molecular structure of the enzyme/4-hydroxybenzoyl-CoA complex was solved by the techniques of multiple isomorphous replacement, solvent flattening, and molecular averaging. Least-squares refinement of the protein model reduced the crystallographic R factor to 18.8% for all measured X-ray data from 30 to 1.8 A resolution. The crystallographic investigation of this dehalogenase revealed that the enzyme is a trimer. Each subunit of the trimer folds into two distinct motifs. The larger, N-terminal domain is characterized by 10 strands of beta-pleated sheet that form two distinct layers which lie nearly perpendicular to one another. These layers of beta-sheet are flanked on either side by alpha-helices. The C-terminal domain extends away from the body of the molecule and is composed of three amphiphilic alpha-helices. This smaller domain is primarily involved in trimerization. The two domains of the subunit are linked together by a cation, most likely a calcium ion. The 4-hydroxybenzoyl-CoA molecule adopts a curved conformation within the active site such that the 4-hydroxybenzoyl and the adenosine moieties are buried while the pantothenate and pyrophosphate groups of the coenzyme are more solvent exposed. From the three-dimensional structure it is clear that Asp 145 provides the side-chain carboxylate group that adds to form the Meisenheimer intermediate and His 90 serves as the general base in the subsequent hydrolysis step. Many of the structural principles derived from this investigation may be directly applicable to other related enzymes such as crotonase.

Computer-assistant prediction of phospholipid binding sites of caldesmon and calponin

The primary structure of smooth muscle caldesmon and calponin was screened for the presence of amphiphilic α-helices which can participate in the formation of protein-lipid contacts. Only one caldesmon segment (residues 645–660) having a predominantly α-helical structure and high hydrophobic moment satisfies all criteria for a surface-seeking helix and is predicted to be involved in the caldesmon-phospholipid interaction. This prediction agrees with experimental results indicating that one of the caldesmon-phospholipid binding sites is located in the sequence 628–658 [Bogatcheva et al. (1994) FEBS Lett. 342, 176]. Two segments of calponin (residues 45–55 and 85–95) exhibit high hydrophobic moments and the sequence 85–95 is characterized by a high probability of α-helix formation. This may suggest that at least one of these segments could facilitate the calponin-phospholipid interaction and that calponin, as with many other actin binding proteins, is able to interact with membranes.

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins

The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and are DD-carboxypeptidases. All three low M r , PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic α-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.

Conformational and binding studies on peptides related to domains I and III of calmodulin

The conformational and ion-binding properties of two peptide fragments of 25 amino acid residues corresponding to the helix-loop sequences of domains I and III of calmodulin (CaM) were investigated by CD and Tb(3+)-mediated fluorescence spectroscopy. Both peptides exhibit very similar ion binding properties either in water or trifluoroethanol (TFE), and do not allow the differentiation of the two domains in the native protein in terms of their binding capacity. An aggregation phenomenon was observed in TFE with increase of the alpha-helical content. We suggest that the aggregation involves an interaction between the hydrophilic surfaces of amphiphilic alpha-helices in a way similar to inverse micelle formation.

Association Characteristics of Amphiphilic α-Helices Connected by Flexible Links

Abstract Amphiphilic helical peptides interlinked with sequences bearing a flexible mobility were designed and synthesized. The sequences containing glycine and/or cystine were adopted for the linkage parts. The secondary structure of the peptides was investigated by circular dichroism(CD) and the super-secondary structure was estimated. The CD spectra and their pH dependence show that the peptides have almost the same α-helix contents as designed and that the helices associate to be “coiled-coil” at neutral pH. The apparent helix contents increase at low pH and some of helices are subject to denaturation at high pH. The denaturation of α-helix bundles by guanidine hydrochloride is contemplated to be three-state transition. It is proposed that the peptides construct α-helix bundles with both loose and tight contacts between helices. Two kinds of folded structure constructed with 2 or more helices are also suggested.