Directing peptide conformation with centrally positioned pre-organized dipeptide segments: studies of a 12-residue helix and beta-hairpin

Secondary structure formation in oligopeptides can be induced by short nucleating segments with a high propensity to form hydrogen bonded turn conformations. Type I/III turns facilitate helical folding while type II'/I' turns favour hairpin formation. This principle is experimentally verified by studies of two designed dodecapeptides, Boc-Val-Phe-Leu-Phe-Val-Aib-Aib-Val-Phe-Leu-Phe-Val-OMe 1 and Boc-Val-Phe-Leu-Phe-Val- (D) Pro- (L) Pro-Val-Phe-Leu-Phe-Val-OMe 2. The N- and C-terminal flanking pentapeptide sequences in both cases are identical. Peptide 1 adopts a largely alpha-helical conformation in crystals, with a small 3(10) helical segment at the N-terminus. The overall helical fold is maintained in methanol solution as evidenced by NMR studies. Peptide 2 adopts an antiparallel beta-hairpin conformation stabilized by 6 interstrand hydrogen bonds. Key nuclear Overhauser effects (NOEs) provide evidence for the antiparallel beta-hairpin structure. Aromatic proton chemical shifts provide a clear distinction between the conformation of peptides 1 (helical) and 2 (beta-hairpin). The proximity of facing aromatic residues positioned at non-hydrogen bonding positions in the hairpin results in extensively ring current shifted proton resonances in peptide 2.

Structural characterization of folded and extended conformations in peptides containing gamma amino acids with proteinogenic side chains: crystal structures of gamma(n), (alpha gamma)(n) and gamma gamma delta gamma sequences

The crystal structures of nine peptides containing gamma(4)Val and gamma(4)Leu are described. The short sequences Boc-gamma(4)(R)Val](2)-OMe 1, Boc-gamma(4)(R)Val](3)-NHMe 2 and Boc-gamma(4)(S)Val-gamma(4)(R)Val-OMe 3 adopt extended apolar, sheet like structures. The tetrapeptide Boc-gamma(4)(R)Val](4)-OMe 4 adopts an extended conformation, in contrast to the folded C-14 helical structure determined previously for Boc-gamma(4)(R)Leu](4)-OMe. The hybrid alpha gamma sequence Boc-Ala-gamma(4)(R)Leu](2)-OMe 5 adopts an S-shaped structure devoid of intramolecular hydrogen bonds, with both alpha residues adopting local helical conformations. In sharp contrast, the tetrapeptides Boc-Aib-gamma(4)(S)Leu](2)-OMe 6 and Boc-Leu-gamma(4)(R)Leu](2)-OMe 7 adopt folded structures stabilized by two successive C-12 hydrogen bonds. gamma(4)Val residues have also been incorporated into the strand segments of a crystalline octapeptide, Boc-Leu-gamma(4)(R)Val-Val-(D)Pro-Gly-Leu-gamma(4)(R)Val-Val-OMe 8. The gamma gamma delta gamma tetrapeptide containing gamma(4)Val and delta(5)Leu residues adopts an extended sheet like structure. The hydrogen bonding pattern at gamma residues corresponds to an apolar sheet, while a polar sheet is observed at the lone delta residue. The transition between folded and extended structures at gamma residues involves a change of the torsion angle from the gauche to the trans conformation about the C-beta-C-alpha bond.

A Disulfide Stabilized beta-Sandwich Defines the Structure of a New Cysteine Framework M-Superfamily Conotoxin

The structure of a new cysteine framework (-C-CC-C-C-C) ``M''-superfamily conotoxin, Mo3964, shows it to have a beta-sandwich structure that is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases outward K+ currents in rat dorsal root ganglion neurons and increases the reversal potential of the Na(V)1.2 channels. The structure of Mo3964 (PDB ID: 2MW7) is constructed from the disulfide connectivity pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for the ``M''-superfamily conotoxins. The tertiary structural fold has not been described for any of the known conus peptides. NOE (549), dihedral angle (84), and hydrogen bond (28) restraints, obtained by measurement of (h3)J(NC') scalar couplings, were used as input for structure calculation. The ensemble of structures showed a backbone root mean square deviation of 0.68 +/- 0.18 angstrom, with 87% and 13% of the backbone dihedral (phi, psi) angles lying in the most favored and additional allowed regions of the Ramachandran map. The conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules.

Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions.

The self-assembly of proteins and peptides into polymeric amyloid fibrils is a process that has important implications ranging from the understanding of protein misfolding disorders to the discovery of novel nanobiomaterials. In this study, we probe the stability of fibrils prepared at pH 2.0 and composed of the protein insulin by manipulating electrostatic interactions within the fibril architecture. We demonstrate that strong electrostatic repulsion is sufficient to disrupt the hydrogen-bonded, cross-β network that links insulin molecules and ultimately results in fibril dissociation. The extent of this dissociation correlates well with predictions for colloidal models considering the net global charge of the polypeptide chain, although the kinetics of the process is regulated by the charge state of a single amino acid. We found the fibrils to be maximally stable under their formation conditions. Partial disruption of the cross-β network under conditions where the fibrils remain intact leads to a reduction in their stability. Together, these results support the contention that a major determinant of amyloid stability stems from the interactions in the structured core, and show how the control of electrostatic interactions can be used to characterize the factors that modulate fibril stability.

A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. (C) 2008 Elsevier Masson SAS. All rights reserved.

Twisting transition between crystalline and fibrillar phases of aggregated peptides

We study two distinctly ordered condensed phases of polypeptide molecules, amyloid fibrils and amyloidlike microcrystals, and the first-order twisting phase transition between these two states. We derive a single free-energy form which connects both phases. Our model identifies relevant degrees of freedom for describing the collective behavior of supramolecular polypeptide structures, reproduces accurately the results from molecular dynamics simulations as well as from experiments, and sheds light on the uniform nature of the dimensions of different peptide fibrils. © 2012 American Physical Society.

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy.

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning, characterization and expression analysis of SIMP (source of immunodominant MHC-associated peptides) in grass carp Ctenopharyngodon idella

SIMP (source of immunodominant MHC-associated peptides) plays a key rote in N-linked glycosylation with the active site of oligosaccharyltransferase, being the source of MHC-peptides in the MHC I presentation pathway. In the present study, the SIMP gene has been cloned from grass carp Ctenopharyngodon idella by rapid amplification of cDNA ends (RACE). The full length of the cDNA sequence is 4384 bp, including a 1117 bp 5' UTR (untranslated region), a 2418 bp open reading frame, and a 849 bp 3' UTR. The deduced amino acids of the grass carp SIMP (gcSIMP) are a highly conserved protein with a STT3 domain and 11 transmembrane regions. The gcSIMP spans over more than 24,212 bp in length, containing 16 exons and 15 introns. Most encoding exons, except the first and the 15th, have the same length as those in human and mouse. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C/EBP beta, and five C/EBP alpha binding sites. The mRNA expression of gcSIMP in different organs was examined by real-time PCR. The gcSIMP was distributed in all the organs examined, with the highest level in brain, followed by the level in the heart, liver, gill, trunk kidney, muscle, head kidney, thymus, and the lowest level in spleen. Furthermore, the recombinant gcSIMP has been constructed successfully and expressed in Escherichia coli by using pQE-40 vector, and the polyclonal antibody for rabbit has been successfully obtained, which was verified to be specific. Identification of gcSIMP will help to explore the function in fish innate immunity. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular and expression characterization of three gonadotropin subunits common alpha, FSH beta and LH beta in groupers

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSH beta and LH beta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSH beta and LH beta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSH beta and LH beta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSH beta and LH beta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSH beta and LH beta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSH beta cells mainly distributed in the middle area of PPD, while the LH beta cells distributed more widely, including in the area similar to the FSH beta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSH beta and LH beta cells. Double immunofluoresence localization further demonstrated FSH beta and LH beta expression in distinct cells in the PPD area, although the FSH beta and LH beta cells were detected in the identical area of PPD. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Characterization of linear epitope of the beta(2)-microglobulin via combination of MALDI-TOF-MS with immunoaffinity

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS), in combination with immunoaffinity provided a powerful tool for determining epitope (antigenic determinant) in protein. The linear epitope of the beta(2)-microglobulin was characterized in the paper. The method as follows: at first beta(2)-microglobulin was digested by a proteolytic enzyme to produce an appropriate set of peptide fragments, then peptide fragments containing the linear epitope were selected and separated from the pool of peptide fragments by immunoprecipitation with the monoclonal antibody. The agarose beads were collected carefully after the reaction. Unbound peptides would be washed away, while the peptides containing the epitope would remain bound to the immobilized antibody after. the beads were washed several times with appropriate buffer. At last the masses of the bound peptides were identified directly by MALDI-TOF MS. Using Endoproteinase Glu-C Endoproteinase Lys-C and Trypsin in the experiment, the linear epitope of beta(2)-microglobulin was located within peptide fragment 59-69, that is, DWSFYLLYYTE.

Level of beta-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure.

BACKGROUND: Heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased level of myocardial betaAR kinase 1 (betaARK1). Our previous studies have shown that inhibition of betaARK1 with the use of the Gbetagamma sequestering peptide of betaARK1 (betaARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of betaARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of betaARK1 inhibition correlates with the degree of heart failure. METHODS AND RESULTS: Transgenic (TG) mice with varying degrees of cardiac-specific expression of betaARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial betaARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of betaARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low betaARKct expression developed severe heart failure, whereas mice with high betaARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of betaARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal betaAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in betaAR function, similar to the WT mice. CONCLUSIONS: These data show that the level of betaARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when betaARK1 inhibition is considered as a molecular target.

Inhibition of beta-adrenergic receptor kinase prevents rapid homologous desensitization of beta 2-adrenergic receptors.

Homologous (agonist-specific) desensitization of beta-adrenergic receptors (beta ARs) is accompanied by and appears to require phosphorylation of the receptors. We have recently described a novel protein kinase, beta AR kinase, which phosphorylates beta ARs in vitro in an agonist-dependent manner. This kinase is inhibited by two classes of compounds, polyanions and synthetic peptides derived from the beta 2-adrenergic receptor (beta 2AR). In this report we describe the effects of these inhibitors on the process of homologous desensitization induced by the beta-adrenergic agonist isoproterenol. Permeabilization of human epidermoid carcinoma A431 cells with digitonin was used to permit access of the charged inhibitors to the cytosol; this procedure did not interfere with the pattern of isoproterenol-induced homologous desensitization of beta 2AR-stimulated adenylyl cyclase. Inhibitors of beta AR kinase markedly inhibited homologous desensitization of beta 2ARs in the permeabilized cells. Inhibition of desensitization by heparin, the most potent of the polyanion inhibitors of beta AR kinase, occurred over the same concentration range (5-50 nM) as inhibition of purified beta AR kinase assessed in a reconstituted system. Inhibition of desensitization by heparin was accompanied by a marked reduction of receptor phosphorylation in the permeabilized cells. Whereas inhibitors of beta AR kinase inhibited homologous desensitization, inhibitors of protein kinase C and of cyclic-nucleotide-dependent protein kinases were ineffective. These data establish that phosphorylation of beta ARs by beta AR kinase is an essential step in homologous desensitization of the receptors. They further suggest a potential therapeutic value of inhibitors of beta AR kinase in inhibiting agonist-induced desensitization.

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy.

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.