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Residue Peptide (residue + peptide)
Selected AbstractsDeamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylaseFEBS JOURNAL, Issue 5 2003Structural, functional implications Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302,6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19,33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15,K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein- l -isoaspartic acid O -methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR,,34 min to ,,31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR,,34 min species decreased with a biphasic time-course with t0.5 -values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of ,,1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial ,-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32,Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition. [source] Differences in the skin peptides of the male and female Australian tree frog Litoria splendidaFEBS JOURNAL, Issue 1 2000The discovery of the aquatic male sex pheromone splendipherin, a new antibiotic peptide caerin 1.10, together with Phe8 caerulein The skin secretions of female and male Litoria splendida have been monitored monthly over a three-year period using HPLC and electrospray mass spectrometry. Two minor peptides are present only in the skin secretion of the male. The first of these is the female-attracting aquatic male sex pheromone that we have named splendipherin, a 25 amino acid peptide (GLVSSIGKALGGLLADVVKSKGQPA-OH). This pheromone constitutes about 1% of the total skin peptides during the breeding season (January to March), dropping to about 0.1% during the period June to November. Splendipherin attracts the female in water at a concentration of 10,11,10,9 m, and is species specific. The second peptide is a wide-spectrum antibiotic of the caerin 1 group, a 25 residue peptide (GLLSVLGSVAKHVLPHVVPVIAEKL-NH2) named caerin 1.10. The neuropeptides of L. splendida are also seasonally variable, the change identical for both the female and male. During the period October to March, the sole neuropeptide present in skin secretions is caerulein [pEQDY(SO3)TGWMDF-NH2]; this is active on smooth muscle and is also an analgaesic. During the southern winter (June to September), more than half of the caerulein is hydrolysed to [pEQDYTGWMDF-NH2], a peptide that shows no smooth muscle activity. In place of caerulein, a new peptide, Phe8 caerulein [pEQDY(SO3)TGWFDF-NH2], becomes a major component of the skin secretion. Perhaps this seasonal change is involved in thermoregulation, that is, with the initiation and maintenance of the inactive (hibernation) phase of the animal. [source] Characterization of conantokin Rl -A: molecular phylogeny as structure/function studyJOURNAL OF PEPTIDE SCIENCE, Issue 8 2010Konkallu H. Gowd Abstract A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl -A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl -A is a 24-residue peptide containing three ,-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl -A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl -A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca++. The order of potency for blocking the four NMDA receptor subtypes by ConRl -A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl -A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. [source] Peptide dynamic fingerprints: a tool for investigating the role of conformational flexibility for GLP-1 analogs affinityJOURNAL OF PEPTIDE SCIENCE, Issue 8 2005Dr M. Adenot Abstract Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide implicated in short-term appetite regulation. Its analogs are presumed to be potential drugs against obesity and non-insulin dependent diabetes mellitus (NIDDM or type 2 diabetes). This study examined how the dynamic fingerprints can be used for establishing dynamics,activity relationships in a series of peptides for which the mechanism of action is unknown and in which mutations can cause an increase or decrease in biological activity. The 3D autocorrelation method was used to generate maps of both active and inactive analogs. As the active conformation of GLP-1 is not yet clearly defined, the dynamic fingerprints of peptides in an aqueous environment were compared to explain the high affinity of the peptide for its receptor. The suggestion that the peptide could bind to the receptor in a folded conformation has been examined. In the case of the GLP-1 analogs, it was shown that the folding tendency cannot be directly related to affinity values and the results do not favor a folded active conformation model of GLP-1. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] ,-Hairpin folding and stability: molecular dynamics simulations of designed peptides in aqueous solutionJOURNAL OF PEPTIDE SCIENCE, Issue 9 2004Clara M. Santiveri Abstract The structural properties of a 10-residue and a 15-residue peptide in aqueous solution were investigated by molecular dynamics simulation. The two designed peptides, SYINSDGTWT and SESYINSDGTWTVTE, had been studied previously by NMR at 278 K and the resulting model structures were classified as 3:5 ,-hairpins with a type I + G1 ,-bulge turn. In simulations at 278 K, starting from the NMR model structure, the 3:5 ,-hairpin conformers proved to be stable over the time period evaluated (30 ns). Starting from an extended conformation, simulations of the decapeptide at 278 K, 323 K and 353 K were also performed to study folding. Over the relatively short time scales explored (30 ns at 278 K and 323 K, 56 ns at 353 K), folding to the 3:5 ,-hairpin could only be observed at 353 K. At this temperature, the collapse to ,-hairpin-like conformations is very fast. The conformational space accessible to the peptide is entirely dominated by loop structures with different degrees of ,-hairpin character. The transitions between different types of ordered loops and ,-hairpins occur through two unstructured loop conformations stabilized by a single side-chain interaction between Tyr2 and Trp9, which facilitates the changes of the hydrogen-bond register. In agreement with previous experimental results, ,-hairpin formation is initially driven by the bending propensity of the turn segment. Nevertheless, the fine organization of the turn region appears to be a late event in the folding process. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] Expediting the Fmoc solid phase synthesis of long peptides through the application of dimethyloxazolidine dipeptidesJOURNAL OF PEPTIDE SCIENCE, Issue 1 2004Dr Peter White Abstract This paper describes the step-wise Fmoc solid phase synthesis of a 95-residue peptide related to FAS death domain. Attempts to prepare this peptide employing conventional amino acid building blocks failed. However, by the judicious use of dimethyloxazolidine dipeptides of serine and threonine, the peptide could be readily prepared in remarkable purity by applying single 1 h coupling reactions. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] N -Terminal domain of HTLV-I integrase.JOURNAL OF PEPTIDE SCIENCE, Issue 11 2001Complexation, conformational studies of the zinc finger Abstract The HTLV-I integrase N -terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9,43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N -terminal domain is likely to be composed of an helical region (residues 28,42) and a ,-strand (residues 20,23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd. [source] The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptideJOURNAL OF PEPTIDE SCIENCE, Issue 4 2001Bengt Erik Haug Abstract Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Why ,2 -antiplasmin must be converted to a derivative form for optimal functionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2007K. N. LEE Summary.,Background:,Human ,2 -antiplasmin (,2AP), the primary inhibitor of fibrinolysis, is secreted from the liver into plasma as a 464-residue protein with Met as the N-terminus. An R6W polymorphism has been suggested to affect fibrinolytic rate. Within circulating blood, antiplasmin-cleaving enzyme (APCE) cleaves Met-,2AP(R6) faster than Met-,2AP(W6) at the Pro12,Asn13 bond to yield Asn-,2AP. Objectives:,To compare Met-,2AP(R6), Met-,2AP(W6) and Asn-,2AP for crosslinking with fibrin and the ability to protect fibrin from digestion by plasmin. Methods and results:,Asn-,2AP utilizes Gln2 (Gln14 in Met-,2AP) to become crosslinked to fibrin approximately twelvefold faster than Met-,2AP(R6) or Met-,2AP(W6), and this enhances the resistance of fibrin to plasmin. All three forms of ,2AP inhibit plasmin at identical rates. The N-terminal 12-residue peptide of Met-,2AP slows crosslinking of Met-,2AP(R6) or Met-,2AP(W6) by limiting access of factor XIIIa to Gln14 rather than shifting crosslinking to other Gln residues. Edman sequencing and mass analyses of tryptic peptides from each ,2AP crosslinked with 5-(biotinamido)pentylamine showed Gln14 as the only major crosslinking site. Residues 5,8, GRQL in Met-,2AP(R6), and residues 1,8, MEPLGWQL in Met-,2AP(W6), slow fibrin crosslinking. Conclusion:,Gln14 in both Met-,2AP(R6) and Met-,2AP(W6) is sheltered by the N-terminal 12-residue peptide, which, when cleaved, yields Asn-,2AP, which is rapidly crosslinked to fibrin and maximally protects it from plasmin. The R6 W polymorphism in Met-,2AP does not affect its crosslinking to fibrin, but it does slow cleavage by APCE and reduces the amount of Asn-,2AP available for rapid crosslinking to fibrin. [source] Analysis of the factors that stabilize a designed two-stranded antiparallel ,-sheetPROTEIN SCIENCE, Issue 6 2002Juan F. Espinosa Abstract Autonomously folding ,-hairpins (two-strand antiparallel ,-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a ,-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in ,-hairpin stability by comparing 1 with DP, N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the DPG segment decreases the entropic cost of ,-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of DPG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed ,-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins. [source] Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspFU -mediated actin assembly and harbours N-terminal sequences that influence pedestal lengthCELLULAR MICROBIOLOGY, Issue 9 2006Kenneth G. Campellone Summary Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) both utilize type III secretion systems that translocate the effector protein Tir into the plasma membrane of mammalian cells in order to stimulate localized actin assembly into ,pedestals'. The Tir molecule that EPEC delivers is phosphorylated within its C-terminus on tyrosine-474, and a clustered 12-residue phosphopeptide encompassing this residue initiates an efficient signalling cascade that triggers actin polymerization. In addition to Y474, tyrosine-454 of EPEC Tir is phosphorylated, although inefficiently, and promotes actin polymerization at low levels. In contrast to EPEC Tir, EHEC Tir lacks Y474 and triggers pedestal formation in a phosphotyrosine-independent manner by interacting with an additional effector protein, EspFU. To identify EHEC Tir sequences that regulate localized actin assembly, we circumvented the strict requirements for type III translocation and directly expressed Tir derivatives in mammalian cells by transfection. Infection of Tir-expressing cells with a Tir-deficient EHEC strain demonstrated that ectopically expressed Tir localizes to the plasma membrane, is modified by mammalian serine-threonine kinases and is fully functional for actin pedestal formation. Removal of portions of the cytoplasmic N-terminus of Tir resulted in the generation of abnormally long pedestals, indicating that this region of EHEC Tir influences pedestal length. In the presence of the entire N-terminal domain, a 12-residue peptide from the C-terminus of EHEC Tir is both necessary and sufficient to recruit EspFU and initiate actin pedestal formation. This peptide encompasses the portion of EHEC Tir analogous to the EPEC Tir-Y454 region and is present within the Tir molecules of all pedestal-forming bacteria, suggesting that this sequence harbours a conserved signalling function. [source] Structure,function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor proteinCHEMICAL BIOLOGY & DRUG DESIGN, Issue 1 2005M. Jaseja Abstract:, Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173,178-residue stretch of the human 37LRP sequence, has been identified as the laminin-1-binding site. Peptides from 37LRP of species that contain this palindrome-bind laminin-1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15-residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin-1-binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin-1-binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin-1-binding affinity of the peptides influences their inhibitory activity. [source] Synthesis and Structural Model of an ,(2,6)-Sialyl-T Glycosylated MUC1 Eicosapeptide under Physiological ConditionsCHEMISTRY - A EUROPEAN JOURNAL, Issue 19 2006Sebastian Dziadek Dr. Abstract To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, a 20-residue peptide (GSTAPPAHGVTSAPDTRPAP) representing the full length tandem repeat sequence of the human mucin MUC1 and its analogue glycosylated with the (2,6)-sialyl-T antigen on Thr11, were prepared and investigated by NMR and molecular modeling. The peptides contain both the GVTSAP sequence, which is an effective substrate for GalNAc transferases, and the PDTRP fragment, a known epitope recognized by several anti-MUC1 monoclonal antibodies. It has been shown that glycosylation of threonine in the GVTSAP sequence is a prerequisite for subsequent glycosylation of the serine at GVTSAP. Furthermore, carbohydrates serve as additional epitopes for MUC1 antibodies. Investigation of the solution structure of the sialyl-T glycoeicosapeptide in a H2O/D2O mixture (9:1) under physiological conditions (25,°C and pH 6.5) revealed that the attachment of the saccharide side-chain affects the conformational equilibrium of the peptide backbone near the glycosylated Thr11 residue. For the GVTSA region, an extended, rod-like secondary structure was found by restrained molecular dynamics simulation. The APDTR region formed a turn structure which is more flexibly organized. Taken together, the joined sequence GVTSAPDTR represents the largest structural model of MUC1 derived glycopeptides analyzed so far. [source] A peptide-based immunoassay for antibodies against botulinum neurotoxin AJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2007M. Zouhair Atassi Abstract Cervical dystonia (CD) is due to neck-muscle spasms that cause pain and involuntary contractions resulting in abnormal neck movements and posture. Symptoms can be relieved by injecting the affected muscle with a botulinum neurotoxin (BoNT, usually type A or type B). The therapeutic benefits are impermanent and toxin injections need to be repeated every 3,6 months. In a very small percentage of patients (less with BoNT/A than with BoNT/B) the treatment elicits blocking anti-toxin antibodies (Abs), which reduce or terminate the patient's responsiveness to further treatment. We have recently mapped (Dolimbek et al., 2006) the CD sera Ab-binding profile using a panel of 60, 19-residue peptides that encompassed the entire H chain sequence 449,1296 and overlapped consecutively by 5 residues. Abs in CD sera bound to one or more of the peptides N25, C10, C15, C20, and C31. This suggested the possibility that binding to these peptides could be used for assay of Abs in CD sera. Data analysis reported here found that Ab binding to these regions showed very significant deviations from the control responses. Of these four peptides, C10 showed the most significant level of separation between patient and control groups (p,=,5,×,10,7) and the theoretical resolution (i.e., ability to distinguish CD patients from control, see full definition under ,Statistical analysis' in Methods), 84%, was about 4% higher than the least resolved response, C31 (p,=,6,×,10,6, resolution 80%). Since the amounts of Abs bound to a given peptide varied with the patient and not all the patients necessarily recognized all four peptides, there was the possibility that binding to combinations of two or more peptides might give a better discriminatory capability. Using two peptides, C10 plus C31, the resolution improved to 87% (p,=,4,×,10,8). These two peptides appeared to compliment each other and negate the lower resolution of C31. Combination of three peptides gave resolutions that ranged from 85 (N25,+,C15,+,C31; p,=,2,×,10,7) to 88% (C10,+,C15,+,C31; p,=,1,×,10,8). Finally, using the data of all four peptides, N25,+,C10,+,C15,+,C31, gave a resolution of 86% (p,=,1,×,10,7). Although these levels of resolution are somewhat lower than that obtained with whole BoNT/A (resolution 97%; p,=,6,×,10,12), it may be concluded that the two-peptide combination C10,+,C31, or the three-peptide combination C10,+,C15,+,C31 (affording resolutions of 87 and 88%, respectively) provide a good diagnostic, toxin-free procedure for assay of total specific anti-toxin Abs in BoNT/A-treated CD patients. Copyright © 2006 John Wiley & Sons, Ltd. [source] |