Cosolvent,ions, and temperature effects on the structural properties of cecropin A‐Magainin 2 hybrid peptide in solutions

Antimicrobial peptides are promising alternative to traditional antibiotics and antitumor drugs for the battle against new antibiotic resistant bacteria strains and cancer maladies. The study of their structural and dynamics properties at physiological conditions can help to understand their stability, delivery mechanisms, and activity in the human body. In this article, we have used molecular dynamics simulations to study the effects of solvent environment, temperature, ions concentration, and peptide concentration on the structural properties of the antimicrobial hybrid peptide Cecropin A–Magainin 2. In TFE/water mixtures, the structure of the peptide retained α‐helix contents and an average hinge angle in close agreement with the experimental NMR and CD measurements reported in literature. Compared to the TFE/water mixture, the peptide simulated at the same ionic concentration lost most of its α‐helix structure. The increase of peptide concentration at both 300 and 310 K resulted in the peptide aggregation. The peptides in the complex retained the initial N‐ter α‐helix segment during all the simulation. The α‐helix stabilization is further enhanced in the high salt concentration simulations. The peptide aggregation was not observed in TFE/water mixture simulations and, the peptide aggregate, obtained from the water simulation, simulated in the same conditions did dissolve within few tens of nanoseconds. The results of this study provide insights at molecular level on the structural and dynamics properties of the CA‐MA peptide at physiological and membrane mimic conditions that can help to better understand its delivery and interaction with biological interfaces. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 1–14, 2015.     

Vasonatrin peptide stimulates both of the natriuretic peptide receptors,NPRA and NPRB

Vasonatrin peptide (VNP) is an active cardiovascular factor and a novel synthetic natriuretic peptide with unknown natriuretic peptide receptor (NPR) binding properties. We set out to design binding models of NPRA/VNP and NPRB/VNP, and then assessed their recognition and binding affinities using molecular dynamics. Molecular dynamics analysis indicated decreases in the values of Van der Waals, electrostatic energy and potential energy of NPRB/VNP compared to NPRA/VNP. There was a 25% increase in H-bond formation between VNP and NPRB. The cGMP stimulated by VNP in NPRB-transfected HEK-293 cells was 11-fold higher than that of NPRA. We therefore demonstrated that VNP binds with both NPRA and NPRB, but with a preference for NPRB.     

Solution, solid phase and computational structures of apicidin and its backbone-reduced analogs.

The recently isolated broad-spectrum antiparasitic apicidin (1) is one of the few naturally occurring cyclic tetrapeptides (CTP). Depending on the solvent, the backbone of 1 exhibits two gamma-turns (in CH(2)Cl(2)) or a beta-turn (in DMSO), differing solely in the rotation of the plane of one of the amide bonds. In the X-ray crystal structure, the peptidic C==Os and NHs are on opposite sides of the backbone plane, giving rise to infinite stacks of cyclotetrapeptides connected by three intermolecular hydrogen bonds between the backbones. Conformational searches (Amber force field) on a truncated model system of 1 confirm all three backbone conformations to be low-energy states. The previously synthesized analogs of 1 containing a reduced amide bond exhibit the same backbone conformation as 1 in DMSO, which is confirmed further by the X-ray crystal structure of a model system of the desoxy analogs of 1. This similarity helps in explaining why the desoxy analogs retain some of the antiprotozoal activities of apicidin. The backbone-reduction approach designed to facilitate the cyclization step of the acyclic precursors of the CTPs seems to retain the conformational preferences of the parent peptide backbone.     

Arsenic contamination of groundwater and prevalence of arsenical dermatosis in the Hetao plain area,Inner Mongolia,China

The present study determined cardiac chamber-specific alterations of the expression of the atrial and brain natriuretic peptide (ANP and BNP) genes with a small increase in age beyond adulthood and with systemic hypertension of intermediate duration. The expression distributions of these genes was determined using in situ hybridization in the right and left atria (RA and LA), and the right and left ventricles (RV and LV) in Wistar Kyoto rats (WKY) and age-matched Spontaneously Hypertensive rats (SHR) at ages 6 months (adult) and 8 months (advanced-age beyond adulthood).In all rat groups, both genes were expressed (ANP > BNP) in the LA and LV, and were not expressed in the RA and RV. The genes were expressed in the LA in all rat groups; the ANP, but not the BNP, expression increased with advancing age and with superimposed hypertension. They were expressed in the LV of the advanced-age WKY, adult and advanced-age SHR, but not in the adult WKY. The ANP mRNA labeling in the LA was diffuse and interspersed with dense accumulations, whereas BNP labeling was diffuse. The labeling of both genes in the form of sparse clusters was seen in the LV of the advanced-age SHR. Our study showed that ANP and BNP expression in left heart chambers increased with a small increase in age, with hypertension of intermediate duration, and with modest left ventricular hypertrophy. The chamber-specific expression distribution could be due to special groups of cardiac cells, or to local chamber-specific factors.     

血管钠肽、 C型钠尿肽和心房钠尿肽舒血管作用的对比

本实验采用离体血管灌流方法,观察和比较血管钠肽（ＶＮＰ）,Ｃ型钠尿肽（ＣＮＰ）和心房钠尿肽（ＡＮＰ）对大鼠肺动脉,腹主动脉和腹腔静脉的舒张作用。．结果表明,ＶＮＰ,ＣＮＰ和ＡＮＰ对离体大鼠的保留内皮与去内皮的肺动脉,腹主动脉和腹腔静脉均有浓度依赖性舒张作用。     

A type-II beta-turn, proline-containing, cyclic pentapeptide as a building block for the construction of models of the cleavage site of pro-oxytocin.

Previous studies have indicated that proteolytic activation of pro-hormones and pro-proteins occurs most frequently at the level of basic amino acids arranged in doublets and that the dibasic sites are situated in or next to beta-turns. Investigations utilizing synthetic peptides reproducing the N-terminal processing domain of pro-oxytocin-neurophysin have suggested a close relationship between the secondary structure of the cleavage locus and enzyme recognition, the minimal recognized sequence being the -Pro-Leu-Gly-Gly-Lys-Arg-Ala-Val-Leu- segment of the native precursor. NMR investigations and energy minimization studies have demonstrated that this sequence is organized in two type-II beta-turns involving the -Pro-Leu-Gly-Gly- and -Lys-Arg-Ala-Val- sequences. To further strengthen the above reported hypothesis and to study the role of turn subtypes, a new proline containing cyclic substrate of the processing enzyme, in which the N-terminal side that comes before the Lys-Arg pair is constrained to adopt a type-lI beta-turn, has been synthesized. The presence of a type-II beta-turn structure in this cyclic peptide model has been demonstrated by a combined NMR, CD and FT-IR absorption investigation. A preliminary study shows that PC1 is able to recognize and process our constrained substrate.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Mechanism,specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases

Signal peptide peptidase (SPP) and the homologous SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 belong to the family of GxGD intramembrane proteases. SPP/SPPLs selectively cleave transmembrane domains in type II orientation and do not require additional co-factors for proteolytic activity. Orthologues of SPP and SPPLs have been identified in other vertebrates, plants, and eukaryotes. In line with their diverse subcellular localisations ranging from the ER (SPP, SPPL2c), the Golgi (SPPL3), the plasma membrane (SPPL2b) to lysosomes/late endosomes (SPPL2a), the different members of the SPP/SPPL family seem to exhibit distinct functions. Here, we review the substrates of these proteases identified to date as well as the current state of knowledge about the physiological implications of these proteolytic events as deduced from in vivo studies. Furthermore, the present knowledge on the structure of intramembrane proteases of the SPP/SPPL family, their cleavage mechanism and their substrate requirements are summarised. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.     

Membrane adsorption,folding, insertion and translocation of synthetic trans-membrane peptides

Spontaneous membrane adsorption, folding and insertion of the synthetic WALP16 and KALP16 peptides was studied by computer simulations starting from completely extended conformations. The peptides were simulated using an unmodified all-atom force field in combination with an efficient Monte Carlo sampling algorithm. The membrane is represented implicitly as a hydrophobic zone inside a continuum solvent modelled using the generalized Born theory of solvation. The method was previously parameterized to match insertion energies of hydrophobic side chain analogs into cyclohexane and no parameters were optimized for the present simulations. Both peptides rapidly precipitate out of bulk solution and adsorb to the membrane surface. Interfacial folding into a helical conformation is followed by membrane insertion. Both the peptide conformations and their location in the membrane are strongly temperature dependent. The temperature dependent behaviour can be summarized by fitting to a four-state model, separating the system into folded and unfolded conformers, which are either inserted into the membrane or located at the interfaces. As the temperature is lowered the dominant peptide conformation of the system changes from unfolded surface bound configurations to folded surface bound states. Folded trans-membrane conformers represent the dominant configuration at low temperatures. The analysis allows direct estimates of the free energies of peptide folding and membrane insertion. In the case of WALP the quality of the fit is excellent and the thermodynamic behaviour is in good agreement with expected theoretical consideration. For KALP the fit is more problematic due to the large solvation energies of the charged lysine residues.     

Identification, structural determination, and biological activity of bovine and canine calcitonin receptor-stimulating peptides

We have recently identified in porcine brain a series of new peptides, designated calcitonin receptor-stimulating peptide-1 (CRSP-1), CRSP-2, and CRSP-3, but failed to find their counterparts in humans and rodents by either database searching or experimental cross-hybridization. In this study, we isolated cDNAs encoding precursors of bovine CRSP-1, canine CRSP-1, and canine CRSP-2 from their thyroid cDNA libraries. Although the deduced mature amino acid sequences of bovine and canine CRSP-1s and canine CRSP-2 showed identity with their respective porcine CRSP counterparts, none of them had a C-terminal amide structure. In LLC-PK(1) cells endogenously expressing the calcitonin (CT) receptor, bovine and canine CRSP-1s enhanced the cAMP production, while canine CRSP-2 did not stimulate it at all. Equine CGRP-I had a high identity in its amino acid sequence with porcine CRSP-1 and stimulated LLC-PK(1) cells at a potency comparable to that of porcine CT. None of these CRSPs or equine CGRP-I stimulated the CT-like receptor, even in the presence of receptor activity-modifying proteins. These results demonstrate that CRSP-1, a new class of biologically active peptide, is present in animals evolutionarily close to pigs and induces its activity through the calcitonin receptor, suggesting a wide existence and common properties of this peptide in mammals.     

The design, synthesis, and characterization of tight-binding inhibitors of calmodulin

Based on a consideration of the probable structure of calmodulin and some natural peptides known to interact with it, two calmodulin-binding peptides were designed. These peptides bind to calmodulin in helical conformations and are capable of forming electrostatic and hydrophobic interactions with calmodulin. Their dissociation constants for binding (less than or equal to 210 and 400 pM) place them as the tightest-binding inhibitors of calmodulin thus far reported. The study of the interactions of these and similar peptides with calmodulin will provide valuable insights into the mechanisms whereby calmodulin binds to target enzymes, and it also serves as an excellent model system for exploring the physical chemistry of protein-protein interaction.     

Purification and Structural Determination of a Phosphorylated Peptide with Anti-calcification and Chitin-binding Activities in the Exoskeleton of the Crayfish,Procambarus clarkii

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anionexchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a RebersRiddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completelyinhibited it at 3×10^-7 M. CAP-1 also showed chitinbinding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.     

Synthesis,conformation, and membrane modifying properties of the trikoningin KB lipopeptaibols: Effect of hydrophobicity and chirality in position 1

Summary We synthesized by solution-phase methods the naturally occurring, 10-amino acid residue lipopeptaibol antibiotics trikoningins KBI and KBII, and the [l-Iva¹] KB analogue, in which the amino acid in position 1 is different, with the aim at investigating the effect of hydrophobicity and chirality in that position. A solution conformational analysis, using FT-IR absorption and CD techniques, indicated that all of the three decapeptides are predominantly helical in a membrane-mimetic environment. Permeability measurements showed an increase of the activity from the [Aib¹] peptide to the more hydrophobic [Iva¹] peptides. Conversely, the effect of a change in chirality, obtained by replacingd-Iva¹ withl-Iva, turned out to be of minor significance.     

The Ribosomal Peptidyl Transferase Center: Structure,Function, Evolution,Inhibition

ABSTRACT

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring riboyzmes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

Amino Acid Sequence and Antimicrobial Activity of Chitin-Binding Peptides,Pp-AMP 1 and Pp-AMP 2, from Japanese Bamboo Shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

家蚕抑前胸腺肽类似物的活性鉴定和结构分析

以家蚕Bombyx mori抑前胸腺肽的氨基酸序列作为基础,通过氨基酸残基的添加、减少和置换,人工合成了一组与家蚕抑前胸腺肽结构类似的多肽。利用家蚕前胸腺体外培养技术,结合蜕皮激素放射免疫分析方法,鉴定了与抑前胸腺肽结构类似的多肽的生理活性,并对它们的活性特征、化学参数、结构和功能、信号传导途径进行了综合的比较和分析。类似物899808的生物学功能与抑前胸腺肽的相同而且活性近似;类似物899805和899809对家蚕前胸腺蜕皮激素的生物合成表现出随浓度增加而增加的促进作用,而低浓度下几乎不促进;899803、899804、899806和899807类似物对家蚕前胸腺蜕皮激素的生物合成的促进和抑制作用与它们的浓度有着依赖关系。实验结果表明,对抑前胸腺肽的氨基酸序列作任何改变,都导致其生理活性的下降、丧失甚至相反的活性。     

Fungal biosynthesis of non-ribosomal peptide antibiotics and alpha, alpha-dialkylated amino acid constituents.

Zervamicins (Zrv) IIA and IIB are membrane modifying peptide antibiotics of fungal origin, characterized by a sequence of 15 amino acid residues. The primary structure of Zrv-IIA contains five alpha-aminoisobutyric acid residues at positions 4, 7, 9, 12 and 14 of the linear peptide. The sequence of Zrv-IIB is similar, but contains a D-isovaline at position 4. When the free amino acid Aib was added to the peptone-glucose culture medium, the fungus Emericellopsis salmosynnemata produced Zrv-IIA as the major secondary metabolite, whereas addition of DL-Iva to the culture led to a high production of Zrv-IIB. This observation is rationalized by a lack of selectivity of the non-ribosomal peptide synthetase with respect to the thiolester activated amino acid substrates during step 12 of peptide synthesis. Analysis of the configuration of the Iva residue of Zrv-IIB showed a high enantiomeric purity of the D-enantiomer, indicating a high stereoselectivity of the peptide synthetase for this substrate.When the culture was supplemented with [(15)N]DL-Iva, the nitrogen isotope was not only found at the D-Iva residue, but surprisingly also at the Aib residues as well as at the proteinogenic residues of Zrv. The partial catabolism of exogenous [(15)N]DL-Iva is explained by the assumption of a decarboxylation-dependent transamination reaction, catalysed by 2,2-dimethylglycine decarboxylase. The same enzyme might also be involved in the reversed carboxylation reactions of acetone and 2-butanone, during the anabolic biosynthesis of Aib and Iva, respectively. Zrv might possibly act as a thermodynamic sink to shift these equilibrium reactions towards the reversed side.     

Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.     

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.