Interactions of the human calcitonin fragment 9-32 with phospholipids: a monolayer study

Human calcitonin and its C-terminal fragment 9-32 (hCT(9-32)) administered in a spray translocate into respiratory nasal epithelium with an effect similar to intravenous injection. hCT(9-32) is an efficient carrier to transfer the green fluorescent protein into excised bovine nasal mucosa. To understand the translocation of hCT(9-32) across plasma membranes, we investigated its interactions with phospholipids and its interfacial structure using model lipid monolayers. A combination of physicochemical methods was applied including surface tension measurements on adsorbed and spread monolayers at the air-water interface, Fourier transform infrared, circular dichroism, and atomic force microscopy on Langmuir-Blodgett monolayers. The results disclose that hCT(9-32) preferentially interacts with negatively charged phospholipids and does not insert spontaneously into lipid monolayers. This supports a nonreceptor-mediated endocytic internalization pathway as previously suggested. Structural studies revealed a random coil conformation of hCT(9-32) in solution, transforming to alpha-helices when the peptide is localized at lipid-free or lipid-containing air-water interfaces. Atomic force microscopy studies of monolayers of the peptide alone or mixed with dioleoylphosphatidylcholine revealed that hCT(9-32) forms filaments rolled into spirals. In contrast, when interacting with dioleoylphosphatidylglycerol, hCT(9-32) does not adopt filamentous structures. A molecular model and packing is proposed for the spiral-forming hCT(9-32).     

Structural investigations of a human calcitonin-derived carrier peptide in a membrane environment by solid-state NMR

Previous studies have shown that human calcitonin (hCT) and its C-terminal fragment hCT(9-32) translocate in nasal epithelium. Moreover, hCT(9-32) was used as a carrier to internalize efficiently the green fluorescent protein, drugs, and plasmid DNA. To understand the mechanism of the membrane crossing process, we determined structural parameters of the carrier peptide hCT(9-32) in a membrane environment using solid-state NMR. For that purpose, we synthesized a multiply labeled hCT(9-32) peptide comprising four positions with fully (15)N- and (13)C-labeled amino acids. Multilamellar vesicle samples containing varying mixing ratios of hCT(9-32) and phospholipids found in the plasma membrane of nasal epithelium were prepared. The typical axially symmetric powder patterns of (31)P NMR spectra confirmed the presence of lamellar bilayers in our samples. The chemical shift anisotropy of the (31)P NMR spectra of the samples in the presence of hCT(9-32) is slightly reduced, revealing weak interaction of the peptide with the lipid headgroups. The peptide does not penetrate the lipid membrane as indicated by very similar (2)H NMR order parameters of the phospholipid fatty acid chains in the absence and presence of the carrier peptide. This membrane topology was confirmed by measurements of paramagnetic enhancement of relaxation rates. The conformation of hCT(9-32) was investigated by cross polarization magic angle spinning NMR methods. All peptide signals were resolved and fully assigned in two-dimensional proton-driven (13)C spin diffusion experiments. The isotropic chemical shifts of (13)CO, (13)Calpha, and (13)Cbeta provide information about the secondary structure of the carrier peptide. The conformation of hCT(9-32) was further corroborated by quantitative phi torsion angle measurements. Two monomeric structural models are consistent with the data: (i) a linear backbone conformation of hCT(9-32) and (ii) an antiparallel beta-sheet structure. These structures are maintained over a wide range of peptide:lipid mixing ratios. No direct indications for fibril formation of hCT(9-32) were found. Dipolar coupling measurements indicate rather high amplitudes of motion of the peptide.     

Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape

Cellular entry of peptide, protein, and nucleic acid biopharmaceuticals is severely impeded by the cell membrane. Linkage or assembly of such agents and cell-penetrating peptides (CPP) with the ability to cross cellular membranes has opened a new horizon in biomedical research. Nevertheless, the uptake mechanisms of most CPP have been controversially discussed and are poorly understood. We present data on two recently developed oligocationic CPP, the sweet arrow peptide SAP, a gamma-zein-related sequence, and a branched human calcitonin derived peptide, hCT(9-32)-br, carrying a simian virus derived nuclear localization sequence in the side chain. Uptake in HeLa cells and intracellular trafficking of N-terminally carboxyfluorescein labeled peptides was studied by confocal laser scanning microscopy and flow cytometry using biochemical markers in combination with quenching and colocalization approaches. Both peptides were readily internalized by HeLa cells through interaction with the extracellular matrix followed by lipid raft-mediated endocytosis as confirmed by reduced uptake at lower temperature, in the presence of endocytosis inhibitors and through cholesterol depletion by methyl-beta-cyclodextrin, supported by colocalization with markers for clathrin-independent pathways. In contrast to the oligocationic SAP and hCT(9-32)-br, interaction with the extracellular matrix, however, was no prerequisite for the observed lipid raft-mediated uptake of the weakly cationic, unbranched hCT(9-32). Transient involvement of endosomes in intracellular trafficking of SAP and hCT(9-32)-br prior to endosomal escape of both peptides was revealed by colocalization and pulse-chase studies of the peptides with the early endosome antigen 1. The results bear potential for CPP as tools for intracellular drug delivery.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

Novel daunorubicin-carrier peptide conjugates derived from human calcitonin segments

Severe and often therapy-limiting side effects are a major obstacle in cancer chemotherapy. New delivery concepts reducing systemic side effects are needed in order to optimize anticancer therapies. Several approaches have been followed, most of them concentrating on macromolecular carriers like liposomes, monoclonal antibodies, serum proteins or polyethylene glycol. We present here a novel type of anthracycline conjugate, using a small carrier peptide derived from the peptide hormone human calcitonin (hCT). The carrier peptide hCT(9-32) has so far been shown to be capable of transporting fluorophores or proteins across cellular membranes. Two different carrier peptide-daunorubicin conjugates were prepared, one with an acid-stable amide bond, the second with an acid-labile hydrazone bond. In vitro studies with daunorubicin linked to the carrier peptide via an acid-labile hydrazone bond demonstrated comparable cytotoxicity to daunorubicin in various daunorubicin sensitive cell lines (neuroblastoma cell lines SK-N-MC and SMS-KAN; HEK 293 T cells). In addition, fluorescence microscopy provided further insight into the mechanism of uptake of the carrier peptide hCT(9-32), indicating that endosomal compartments with reduced pH are involved in the intracellular release of daunorubicin.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of ζ-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

Calcitonin-derived carrier peptide plays a major role in the membrane localization of a peptide-cargo complex

Bilayers made of dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) mixture containing or not cholesterol (Chl) were used to investigate the interaction of a carrier peptide with membranes. Atomic force microscopy revealed that the C-terminal 9-32 fragment of human calcitonin (hCT (9-32)), free or coupled to enhanced green fluorescent protein (hCT-eGFP) cargo forms aggregates in the DOPC fluid phase in absence of Chl and in the DPPC enriched liquid-ordered phase when Chl is present. The data show that hCT (9-32) plays a determinant role in the membrane localization of the peptide-cargo complex. They suggest that carpet-like mechanism for membrane destabilization may be involved in the carrier function of hCT (9-32).     

Biophysical and biological studies of end-group-modified derivatives of Pep-1

Pep-1 is a tryptophane-rich cell-penetrating peptide (CPP) that has been previously proposed to bind protein cargoes by hydrophobic assembly and translocate them across cellular membranes. To date, however, the molecular mechanisms responsible for cargo binding and translocation have not been clearly identified. This study was conducted to gain insight into the interaction between Pep-1 with its cargo and the biological membrane to identify the thereby involved structural elements crucial for translocation. We studied three peptides differing in their N- and C-termini: (i) Pep-1, carrying an acetylated N-terminus and a C-terminal cysteamine elongation, (ii) AcPepWAmide, with an acetylated N-terminus and an amidated C-terminus, and (iii) PepW, with two free termini. Thioredoxin (TRX) and beta-galactosidase were used as protein cargoes. To study CPP-membrane interactions, we performed biophysical as well as biological assays. To mimic biological membranes, we used phospholipid liposomes in a dye leakage assay and surfactant micelles for high-resolution NMR studies. In addition, membrane integrity, cell viability, and translocation efficiency were analyzed in HeLa cells. An alpha-helical structure was found for all peptides in the hydrophobic N-terminal region encompassing residues 4-13, whereas the hydrophilic region remained unstructured in the presence of micelles. Our results show that the investigated peptides interacted with the micelles as well as with the protein cargo via their tryptophan-rich domain. All peptides displayed an orientation parallel to the micelle surface. The C-terminal cysteamine group formed an additional membrane anchor, leading to more efficient translocation properties in cells. No membrane permeabilization was observed, and our data were largely compatible with an endocytic pathway for cellular uptake.     

Cover Image

Human calcitonin (hCT) is a 32-residue peptide hormone that is produced in the thyroid where it regulates blood calcium levels and promotes bone formation. While hCT was previously used to treat bone disorders, this has ceased because hCT has been shown to aggregate when elevated levels of the peptide are either produced naturally or injected, and the resulting amyloid fibrils toxic. Deguo Du and his team report the effects of cholesterol, cholesterol sulfate and 3β-[N- (dimethylaminoethane) carbamoyl]- cholesterol (DC-cholesterol) on oxidised hCT, revealing an inhibitory effect of DC-cholesterol on amyloidogenesis. The results provide mechanistic information on the interactions at play and may aid the design of steroid-based structures for inhibiting hCT aggregation. (DOI: 10.1002/bip.23343)

     

NMR characterization of monomeric and oligomeric conformations of human calcitonin and its interaction with EGCG

Calcitonin is a 32-residue peptide hormone known for its hypocalcemic effect and its inhibition of bone resorption. While calcitonin has been used in therapy for osteoporosis and Paget's disease for decades, human calcitonin (hCT) forms fibrils in aqueous solution that limit its therapeutic application. The molecular mechanism of fiber formation by calcitonin is not well understood. Here, high-resolution structures of hCT at concentrations of 0.3 mM and 1 mM have been investigated using NMR spectroscopy. Comparing the structures of hCT at different concentrations, we discovered that the peptide undergoes a conformational transition from an extended to a β-hairpin structure in the process of molecular association. This conformational transition locates the aromatic side chains of Tyr12 and Phe16 in a favorable way for intermolecular π-π stacking, which is proposed to be a crucial interaction for peptide association and fibrillation. One-dimensional (1)H NMR experiments confirm that oligomerization of hCT accompanies the conformational transition at 1 mM concentration. The effect of the polyphenol epigallocatechin 3-gallate (EGCG) on hCT fibrillation was also investigated by NMR and electron microscopy, which show that EGCG efficiently inhibits fibril formation of hCT by preventing the initial association of hCT before fiber formation. The NMR experiments also indicate that the interaction between aromatic rings of EGCG and the aromatic side chains of the peptide may play an important role in inhibiting fibril formation of hCT.     

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.     

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an alpha helical conformation between the Ile4 to Leu12 or to Ala15 region. Simulated annealing showed that the helical region extends from Ile4 to Met19. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.     

High expression of a recombinant human calcitonin precursor peptide in Escherichia coli

Human calcitonin (hCT) is a C-terminus -amidated peptide hormone consisting of 32 amino acids. The amidated structure is essential for its biological activities, and the C-terminal-glycine-extended precursor peptide, hCT[G], is converted to bioactive hCT by a C-terminus--amidating enzyme. An efficient production method is described for the hCT[G] peptide, as a part of the fusion protein consisting of a modified E. coli -galactosidase, linker amino acids and hCT[G]. Stable inclusion bodies of the fusion protein in E. coli were expressed by focusing on the amino acid charge, and the fusion protein was modified by inserting a basic amino acid sequence into its linker region. This modification greatly affected the formation of inclusion bodies. E. coli strain W3110/pG97S4DhCT [G]R4 could produce a large amount of stable inclusion bodies, and the hCT[G] peptide was released quantitatively from the fusion protein by S. aureus V8 protease. This enabled a large-scale production method to be established for the hCT[G] precursor peptide in E. coli to produce mature hCT.     

Structure, dynamics, and antimicrobial and immune modulatory activities of human LL-23 and its single-residue variants mutated on the basis of homologous primate cathelicidins

LL-23 is a natural peptide corresponding to the 23 N-terminal amino acid residues of human host defense cathelicidin LL-37. LL-23 demonstrated, compared to LL-37, a conserved ability to induce the chemokine MCP-1 in human peripheral blood mononuclear cells, a lack of ability to suppress induction of the pro-inflammatory cytokine TNF-α in response to bacterial lipopolysaccharides (LPS), and reduced antimicrobial activity. Heteronuclear multidimensional nuclear magnetic resonance (NMR) characterization of LL-23 revealed similar secondary structures and backbone dynamics in three membrane-mimetic micelles: SDS, dodecylphosphocholine (DPC), and dioctanoylphosphatidylglycerol. The NMR structure of LL-23 determined in perdeuterated DPC contained a unique serine that segregated the hydrophobic surface of the amphipathic helix into two domains. To improve our understanding, Ser9 of LL-23was changed to either Ala or Val on the basis of homologous primate cathelicidins. These changes made the hydrophobic surface of LL-23 continuous and enhanced antibacterial activity. While identical helical structures did not explain the altered activities, a reduced rate of hydrogen-deuterium exchange from LL-23 to LL-23A9 to LL-23V9 suggested a deeper penetration of LL-23V9 into the interior of the micelles, which correlated with enhanced activities. Moreover, these LL-23 variants had discrete immunomodulatory activities. Both restored the TNF-α dampening activity to the level of LL-37. Furthermore, LL-23A9, like LL-23, maintained superior protective MCP-1 production, while LL-23V9 was strongly immunosuppressive, preventing baseline MCP-1 induction and substantially reducing LPS-stimulated MCP-1 production. Thus, these LL-23 variants, designed on the basis of a structural hot spot, are promising immune modulators that are easier to synthesize and less toxic to mammalian cells than the parent peptide LL-37.     

Developing novel hCT derived cell-penetrating peptides with improved metabolic stability

Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9-32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9-32) derivatives by replacing positions 12 and/or 16 of hCT(9-32) with either N-methylphenylalanine or d-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9-32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.     

Developing novel hCT derived cell-penetrating peptides with improved metabolic stability

Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9-32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9-32) derivatives by replacing positions 12 and/or 16 of hCT(9-32) with either N-methylphenylalanine or d-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9-32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.     

The interaction of beta-amyloid protein fragment (12-28) with lipid environments.

The neurotoxicity of beta-amyloid protein (beta AP) fragments may be a result of their solution conformation, which is very sensitive to solution conditions. In this work we describe NMR and CD studies of the conformation of beta AP(12-28) in lipid (micelle) environments as a function of pH and lipid type. The interaction of beta AP(12-28) with zwitterionic dodecylphosphocholine (DPC) micelles is weak and alters the conformation when compared to water solution alone. By contrast, the interaction of the peptide with anionic sodium dodecylsulfate (SDS) micelles is strong: beta AP(12-28) is mostly bound, is alpha-helical from K16 to V24, and aggregates slowly. The pH-dependent conformation changes of beta AP(12-28) in solution occur in the pH range at which the side-chain groups of E22, D23, H13, and H14 are deprotonated (pKas ca. 4 and 6.5); the interaction of beta AP(12-28) with SDS micelles alters the pH-dependent conformational transitions of the peptide whereas the weak interaction with DPC micelles causes little change.     

Solution structure of the antimicrobial peptide gaegurin 4 by H and 15N nuclear magnetic resonance spectroscopy.

Gaegurin 4 (GGN4) is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an alpha-helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic alpha-helices extending from residues 2-10 and from residues 16-32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix-assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C-terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.     

Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation

Human calcitonin (hCT) is a 32-residue peptide that aggregates to form amyloid fibrils under appropriate conditions. In this study, we investigated the effect of the intramolecular disulfide bond formed at the N-terminal region of the peptide in the aggregation kinetics of hCT. Our results indicate that the presence of the disulfide bond in hCT plays a crucial role in forming the critical nucleus needed for fibril formation, facilitating the rate of hCT amyloidogenesis. Furthermore, we reported for the first time the effects of cholesterol, cholesterol sulfate, and 3β-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol) on the amyloid formation of oxidized hCT. Our results show that while cholesterol does not affect amyloidogenesis of oxidized hCT, high concentrations of cholesterol sulfate exhibits a moderate inhibiting activity on hCT amyloid formation. In particular, our results show that DC-cholesterol strongly inhibits amyloidogenesis of oxidized hCT in a dose-dependent manner. Further studies at different pH conditions imply the crucial impact of electrostatic and hydrogen bonding interactions in mediating the interplay of hCT and the surface of DC-cholesterol vesicles and the inhibiting function of DC-cholesterol on hCT fibrillization.     

pH-Dependent conformational changes and topology of a herpesvirus translocating peptide in a membrane-mimetic environment

Pol peptide, an oligopeptide corresponding to the 27 C-terminal amino acids of DNA polymerase from herpes simplex virus type 1, has recently been suggested to translocate from endosomal compartments into the cytosol after being intracellularly delivered via a protein carrier. While an acidic environment was thought to be important for Pol peptide membrane translocation, the mechanism of translocation remains unclear. To investigate the influence of an acidic environment on the conformational properties of the peptide and on its propensity to interact with lipid bilayers, we characterized the structure of Pol peptide at different pH values by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The influence of detergent micelles, which mimic biological lipid membranes, on the peptide secondary structure was also studied. Our CD results indicate that the peptide is in a random conformation in aqueous solution at both acidic and basic pH, whereas in the presence of dodecylphosphocholine (DPC) micelles, it assumes a partial alpha-helical structure which is significantly pH-dependent. An NMR study confirmed that, in the presence of DPC micelles, a short C-terminal alpha-helix is present at pH 6.5, whereas almost two-thirds of the peptide (residues 10-26) fold into an extended amphipathic alpha-helix at pH 4.0. The orientation of Pol peptide relative to the DPC micelle was investigated using paramagnetic probes at both pH 4.0 and 6.5. These studies show that the peptide inserts deeply into the micelle at pH 4.0, whereas it is more exposed to the aqueous environment at pH 6.5. On the basis of these results, a model which might explain the mechanism of translocation of Pol peptide from acidic endosomes to the cytosol is discussed.