Solution conformation of N-terminal fragments of trichosanthin small domain (TCS 182-200)

Three peptides, T14, T18 and TDK, derived from the N-terminus of trichosanthin small domain (TCS 182-200) have been investigated by circular dichroism. Secondary structure and structural transitions of the above peptides under different conditions were studied. Alcohol prompts a transition of the T18 peptide from a β-sheet to an α-helical structure. It also increases the α-helicities of T14 and TDK. The β-sheet of T18 peptide appears more hydrophobic than the α-helix of T14 or TDK. The effects of polypeptide sequence and solvent on secondary structure formation of these model peptides are discussed.     

Design and synthesis of amphipathic antimicrobial peptides

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic α-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no α-helicity in buffer or liposomes, exhibits a modest degree of α-helical structure in the presence of the a-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an α-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.     

膜活性多肽(MAPs)的抗菌活性及其作用机制的研究进展

膜活性多肽(MAPs)是一类具有较好抗菌活性的抗菌肽。作为先天宿主防御分子,广泛的分布于细菌、植物、无脊椎动物、脊椎动物中。膜活性多肽具有抗菌肽的结构特征,肽链通常较短,带正电荷,具有两亲性的α-螺旋或β-折叠结构,通过破坏膜的通透性杀死细菌、真菌和部分肿瘤细胞。膜活性多肽在细胞膜或细胞内部存在特定的分子靶点,并因其独特的作用机制而成为一种新型的肽类抗生素。本文主要对膜活性多肽的抗菌活性及其作用机制的研究现状和发展情况做一综述。     

Surface-induced conformational switching in amphiphilic peptide segments of apolipoproteins B and E and model peptides†

The conformational and surface-binding properties of a synthetic peptide corresponding to Tyr-apolipoprotein B-100(1000–1016) amide, SP-4, which was previously shown to mimic the focal accumulation pattern of LDL on the healing de-endothelialized rabbit aorta [Shih et al. (1990) Proc. Natl. Acad. Sci. USA 87 , 1436–1440], have been investigated. SP-4 behaves as an amphiphilic α-helical peptide at the air-water interface and bound to siliconized quartz slides. However, its N^α-acetylated analogue formed β-sheet structures at the air-water interface. Nonhomologous peptide models of SP-4 also exhibited mixed α-helical and β-sheet surface-binding behavior. Peptides corresponding to the cationic apolipoprotein (apo) B/E receptor binding regions of apoE (SP-2) and apoB (SP-11) were also studied. SP-2 behaved as an amphiphilic α helix, but, surprisingly, SP-11 formed surface-induced β-sheets. These results demonstrate that all of the peptides studied have surface-binding properties, and suggest further that either α-helical or β-sheet peptide structures may determine the binding of LDL to the arterial wall or the apoB/E receptor.     

Conformation of a water-soluble β-sheet model peptide. A circular dichroism and Fourier-transform infrared spectroscopie study of double D-amino acid replacements

Among peptide secondary structures β-sheet domains have been much less intensively studied than α-helical conformations, mainly because of the lack of well characterized model peptides. In the present paper the secondary structure of a water-soluble de novo peptide consisting of 26 amino acids (DPKGDPKGVTVTVTVTVTGKGDPKPD-NH₂) and the corresponding double D-amino acid replacement set have been studied by circular dichroism and Fourier-transform infrared spectroscopy. The model peptide was found to be unstructured in aqueous solution at peptide concentrations < 10^?3 mol/L but to adopt a predominantly β-sheet structure in the presence of 15 mM sodium dodecyl sulfate or at apolar/water interfaces. Although the peptide is composed of amino acids with low helical propensity, it formed a single-stranded helical structure in aqueous trifluoroethanol. The D-amino acid replacement set was synthesized in order to study the conformational stability of the model peptide selectively in distinct regions. The data show that both the α-helix present in 50% trifluoroethanol as well as the β-sheet domain formed in the presence of sodium dodecyl sulfate or at apolar/water interfaces, are located in the region between Val⁹ and Thr¹⁸. Pairwise substitution of adjacent amino acids by their corresponding D-amino acids provides a pronounced β-sheet disturbance. These findings demonstrate that double D-amino acid replacements may be used to locate β-sheet domains in peptides.     

Comparison of the binding of α-helical and β-sheet peptides to a hydrophobic surface

The induction and stabilisation of secondary structure for a series of amphipathic α-helical and β-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from β-sheet to α-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85°C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.     

Synthesis and secondary structural studies of penta(acetyl-Hmb)Aβ(1–40)

Abstract: The Fmoc solid phase synthesis of Aβ(1–40), a strongly aggregating peptide found in Alzheimer’s disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N^α-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using ‘semi-on-line’ MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N-dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl-Hmb-protected and native Aβ(1–40) were performed to investigate the structural basis of Hmb-mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of Aβ(1–40). Despite the reported structure-breaking activity of Hmb groups, penta(acetyl-Hmb)Aβ(1–40) was found to adopt both α-helix and intermolecular β-sheet conformations. In 100% TFE a mixed α-helix/random coil structure was formed by the protected peptide indicating reduced α-helical propensity relative to Aβ(1–40). The protected peptide formed β-sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native Aβ(1–40), penta(acetyl-Hmb)Aβ(1–40) did not form large aggregate species.     

Design,synthesis and characterization of antimicrobial pseudopeptides corresponding to membrane-active peptide

Abstract: To obtain active and metabolically stable analogues, peptide backbone modifications have been incorporated into many biologically active peptides. In this study, we designed and synthesized pseudopeptides corresponding to the antimicrobial peptide that acted on the lipid membrane of the pathogen. Most pseudopeptides exhibited a longer half-life than the peptide in the presence of serum as well as a considerable activity against test bacteria and fungi. Circular dichroism spectra and retention times of the pseudopeptides helped us to elucidate the effect of the incorporation of backbone modifications on the structural parameters necessary for the activity, indicating that α-helical structure was the most important factor for the activity and hydrophobicity had a considerable effect on the activity. Backbone modifications employed in this study can be a useful tool for structure–activity relationship studies and the development of therapeutic agents from membrane-active antimicrobial peptides.     

重组HPV病毒样颗粒二级结构的圆二色谱法预测

目的 分析重组HPV病毒样颗粒（virus-like particle,VLP）的二级结构。方法 取重组HPV16、18、52、58型VLP各1批,均调整蛋白浓度至250〜500 μg/ml,采用圆二色谱远紫外扫描,预测重组HPV VLP的二级结构。结果 重组HPV16 VLP二级结构的预测结果为α螺旋11.7%,反平行β折叠37.1%,平行β折叠4.5%,β转角19.2%,不规则卷曲30.3%;重组HPV18 VLP二级结构的预测结果为α螺旋11.9%,反平行β折叠36.9%,平行β折叠4.6%,β转角19.0%,不规则卷曲30.7%;重组HPV52 VLP二级结构的预测结果为α螺旋12.3%,反平行β折叠37.1%,平行β折叠4.7%,β转角19.0%,不规则卷曲30.2%;重组HPV58 VLP二级结构的预测结果为α螺旋11.7%,反平行β折叠35.3%,平行β折叠4.4%,β转角20.4%,不规则卷曲31.3%。结论 圆二色谱法预测了重组HPV16/18/52/58型VLP的二级结构,该技术可用于重组HPV VLP的结构确证研究。     

Influence of preformed α-helix and α-helix induction on the activity of cationic antimicrobial peptides

One prominent class of cationic antibacterial peptides comprises the α-helical class, which is unstructured in free solution but folds into an amphipathic α-helix upon insertion into the membranes of target cells. To investigate the importance of α-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form α-helices or to have preformed α-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i+ 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized α-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of α-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase α-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r²= 0.88) with a helican parameter which was calculated as the induction of α-helix in α membrane-mimicking environment divided by the α-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.     

Design of potent, non-toxic anticancer peptides based on the structure of the antimicrobial peptide, temporin-1CEa

Recent advances in the search for novel anticancer agents have indicated that the positively charged antimicrobial peptides have emerged as promising agents offering several advantages over the conventional anticancer drugs. As a naturally occurring, cationic, α-helical antimicrobial peptide, temproin-1CEa has been proved to exhibit a potent anticancer effect and a moderate hemolytic activity. In order to reduce the hemolytic activity of temporin-1CEa and improve its anticancer potency towards a range of human breast cancer cells, in the present study, six analogs of temporin-1CEa were rationally designed and synthesized. The amphipathicity levels and α-helical structural patterns of peptides were reserved, while their cationic property and hydrophobicity were changed. The results of MTT and hemolysis assay indicated that the analog peptides displayed an improved anticancer activity and showed an overall optimized therapeutic index. The hydrophobicity of peptides was positively correlated with their hemolytic and antitumor activities. Moreover, the data suggest a strategy of increasing the cationicity while maintaining the moderate hydrophobicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells such as human erythrocytes. This work highlights the potential for rational design and synthesis of improved antimicrobial peptides that have the capability to be used therapeutically for treatment of cancers.     

In silico analysis of antifungal peptides

Antimicrobial peptides are short peptides (< 100 amino acids) that have a potent function against microbial invasion. These peptides are produced by various organisms, including bacteria, fungi, flowering and non-flowering plants, insects and mammals. Antifungal peptides are a major group of antimicrobial peptides that have a specially potent effect against fungi. Several parameters affect the activity of antifungal peptides, including the sequence, size, charge, degree of structure formation, cationicity, hydrophobicity and amphipathicity. By analysis of numerous antifungal peptide sequences, the roles of these parameters in the structure of antifungal peptides are investigated in this review and by the in silico analysis of the existing residues, occupying each position of sequence, a template sequence is defined to generate potent and efficient lead antifungal peptides.     

Synthesis and solubility properties of peptide fragments of human hemoglobin α-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin α-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a <P_C > value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that β-sheet structure played an important role in the insolubility of peptides. When a peptide has a β-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

Probing the α-Helical Structural Stability of Stapled p53 Peptides: Molecular Dynamics Simulations and Analysis

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein–protein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled α-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted α-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute α-helical stability. These simulations provide new insights into the design of α-helical stapled peptides and the development of potent inhibitors of α-helical protein–protein interfaces.     

Electrostatic effects on the α-helix and β-strand formation of BPTI(16–36) studied by Monte Carlo simulated annealing

Abstract: The electrostatic effects on the secondary structure forming tendencies of a peptide fragment with residues 16–36 of bovine pancreatic trypsin inhibitor, BPTI( 16 - 34 ), are studied using Monte Carlo simulated annealing simulations. We consider three dielectric functions ε(r) of distance r: constant dielectric function (ε = 2; strong electrostatic interactions) and sigmoidal functions varying from ε (0) = 2 to ε(∞) = 47 (intermediate) and to ε(∞) = 78 (weak). Simulations with ε = 2 suggest that this peptide exhibits a significant propensity for β-strand formations in accordance with a β-sheet structure of the relevant segment in native BPTI. The tendency for α-helix formations becomes almost comparable with that of β-strands in the simulation with ε(∞) = 47, and there appears no appreciable conformational propensity for this case. Finally, the results with ε(∞) = 78 generate low-energy conformations with conspicuous α-helices. These findings suggest the possibility that the change in electrostatic interactions can be the key factor for the conformational transitions of peptides between α-helix and β-sheet that have recently been observed in experiments. These changes in electrostatic interactions can arise from those in various environmental factors such as conformations of the rest of the protein molecule and solvent conditions.     

Structure of a paralytic peptide from an insect,Manduca sexta

Abstract: Paralytic peptide 1 (PP1) from a moth, Manduca sexta, is a 23-residue peptide (Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu-Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe) that was first found to have paralytic activity when injected into M. sexta larvae. Recent studies demonstrated that PP1 also stimulated the spreading and aggregation of a blood cell type called plasmatocytes and inhibited bleeding from wounds. We determined the solution structure of PP1 by two-dimensional¹H NMR spectroscopy to begin to understand structural–functional relationships of this peptide. PP1 has an ordered structure, which is composed of a short antiparallel β-sheet at residues Tyr¹¹-Thr¹⁴ and Arg¹⁸-Pro²¹, three β turns at residues Phe³-Gly⁶, Ala⁸-Tyr¹¹ and Thr¹⁴-Gly¹⁷, and a half turn at the carboxyl-terminus (residues Lys²⁰-Phe²³). The well-defined secondary and tertiary structure was stabilized by hydrogen bonding and side-chain hydrophobic interactions. In comparison with two related insect peptides, whose structures have been solved recently, the amino-terminal region of PP1 is substantially more ordered. The short antiparallel β-sheet of PP1 has a folding pattern similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF). Therefore, PP1 may interact with EGF receptor-like molecules to trigger its different biological activities.     

1H-NMR signal assignments and secondary structure analysis of martentoxin

Martentoxin is a peptide of 37 amino acid residues purified from the venom of the Chinese scorpion Buthus martensi Karch, which has been demonstrated to be an inhibitor of voltage-dependent sodium channel and voltage-dependent delayed rectifier potassium channel. To elucidate the molecular mechanism of this interaction, the structure of martentoxin was studied by 2D-NMR. The secondary structure of martentoxin consists of a triple-stranded β-sheet connected to a α-helical structure. This helix encompasses 10 residues from Ser11 to Lys20. The three strands of β-sheet probably comprise residues Gly2-Asp5, Q27-N30 and Glu33-Cys36, Cys30-Asn33 with a type I′β turn centered on Asn31-Asn32. The results indicate that martentoxin possesses the conserved β α β β structure of all the potassium channel toxins.     

Identification, synthesis and characterization of a novel antimicrobial peptide HKPLP derived from Hippocampus kuda Bleeker

A novel gene encoding 55 amino-acid residues has been identified from the brooding pouch cDNA library of Hippocampus kuda Bleeker. The deduced amino-acid sequence is highly homologous to several pleurocidin-like peptides from the winter flounder and comprises a signal peptide, a pro-peptide and a mature peptide. The glycine-rich mature peptide, designated HKPLP, contains 24 amino-acid residues and has been synthesized by solid-phase peptide synthesis. The purified HKPLP exhibits antimicrobial activity against several Gram-positive and Gram-negative bacterial strains at low concentrations (MIC 1.5-7.5?μM). Thermal stability assay data show good heat stability. CD spectroscopy experiments indicate that the dominant contents are anti-parallel and parallel sheets, which may have β-sheet or β-strand motif. It is inferred that HKPLP participates in the host defense during egg fertilization and embryo development as an antimicrobial peptide in brooding pouch.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences,model membrane binding and biological activities

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt β-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.