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Peptide Bonds (peptide + bond)
Terms modified by Peptide Bonds Selected AbstractsChemInform Abstract: Nucleophilic Carbene and HOAt Relay Catalysis in an Amide Bond Coupling: An Orthogonal Peptide Bond Forming Reaction.CHEMINFORM, Issue 14 2008Harit U. Vora Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Conformational Transition in the Aminoacyl t-RNA Site of the Bacterial Ribosome both in the Presence and Absence of an Aminoglycoside AntibioticCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2007Samy O. Meroueh Peptide bonds are made at the ribosomal decoding site. Structural information reveals that two bases in the RNA that constitute the decoding site, A1492 and A1493, can have both intrahelical and extrahelical conformations. Aminoglycoside antibiotics bind to the decoding site, and the structural information reveals the two bases in the extrahelical positions. We have shown by explicit-solvent molecular dynamics simulations and free-energy calculations that ribosomal RNA bases A1492 and A1493 are inherently prone to sampling conformational states that include both intrahelical and extrahelical positions. The simulations reveal that base flipping occurs through the minor groove of the double helix. Furthermore, free-energy calculations for the conformational change of the bases to the extrahelical positions in both processes are exergonic and highly favorable. It is likely that the correct codon-anticodon recognition by mRNA and tRNA arrests the bases in extrahelical conformations in the course of normal translation. In contrast, the sequestration of the aminoglycoside antibiotic at the decoding site facilitates the conformational change of the bases to the extrahelical position. Once the antibiotic is bound, the extrahelical positions for the bases are highly favored based on contributions by both electrostatic and entropic components of the free energy for the process. [source] Thrombin-mediated impairment of fibroblast growth factor-2 activityFEBS JOURNAL, Issue 12 2009Pierangela Totta Thrombin generation increases in several pathological conditions, including cancer, thromboembolism, diabetes and myeloproliferative syndromes. During tumor development, thrombin levels increase along with several other molecules, including cytokines and angiogenic factors. Under such conditions, it is reasonable to predict that thrombin may recognize new low-affinity substrates that usually are not recognized under low-expression levels conditions. In the present study, we hypothesized that fibroblast growth factor (FGF)-2 may be cleaved by thrombin and that such action may lead to an impairment of its biological activity. The evidence collected in the present study indicates that FGF-2-induced proliferation and chemotaxis/invasion of SK-MEL-110 human melanoma cells were significantly reduced when FGF-2 was pre-incubated with active thrombin. The inhibition of proliferation was not influenced by heparin. Phe-Pro-Arg-chloromethyl ketone, a specific inhibitor of the enzymatic activity of thrombin, abolished the thrombin-induced observed effects. Accordingly, both FGF-2-binding to cell membranes as well as FGF-2-induced extracellular signal-regulated kinase phosphorylation were decreased in the presence of thrombin. Finally, HPLC analyses demonstrated that FGF-2 is cleaved by thrombin at the peptide bond between residues Arg42 and Ile43 of the mature human FGF-2 sequence. The apparent kcat/Km of FGF-2 hydrolysis was 1.1 × 104 m,1·s,1, which is comparable to other known low-affinity thrombin substrates. Taken together, these results demonstrate that thrombin digests FGF-2 at the site Arg42-Ile43 and impairs FGF-2 activity in vitro, indicating that FGF-2 is a novel thrombin substrate. [source] Imine Additions of Internal Alkynes for the Synthesis of Trisubstituted (E)-Alkene and Cyclopropane Peptide IsosteresADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-13 2005Peter Wipf Abstract Divergent multi-component reactions (DMCR) involving CC bond formations can provide large increases in structural diversity and allow the rapid assembly of complex products from readily available starting materials. Cascade hydrozirconation-Zr/Zn transmetalation-imine addition of alkynes represents a versatile methodology for the synthesis of (E)-alkene and cyclopropane dipeptide isosteres. Appropriate substitutions at the sp2 -carbon of (E)-alkene peptide isosteres allow a range of Pd-catalyzed cross-coupling reactions, which can be used for the fine-tuning of the conformational and electronic properties of the parent peptide bond mimic. CC bond formation by microwave-accelerated Stille coupling of stannylalkenes represents a fast, convergent synthetic approach toward trisubstituted (E)-alkene dipeptide isosteres. [source] Quantum mechanical study of the conformational behavior of proline and 4R-hydroxyproline dipeptide analogues in vacuum and in aqueous solutionJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 3 2002Caterina Benzi The conformational behavior of the title compounds has been investigated by Hartree,Fock, MP2, and DFT computations on the most significant structures related to variations of the backbone dihedral angles, cis/trans isomerism around the peptide bond, and diastereoisomeric puckering of the pyrrolidine ring. In vacuum the reversed , turn (,l), characterized by an intramolecular hydrogen bridge, corresponds to the absolute energy minimum for both puckerings (up and down) of the pyrrolidine ring. An additional energy minimum is found in the helix region, but only for an up puckering of the pyrrolidine ring. When solvent effects are included by means of the polarizable continuum model the conformer observed experimentally in condensed phases becomes the absolute minimum. The down puckering is always favored over its up counterpart, albeit by different amounts (0.4,0.5 kcal/mol for helical structures and about 2 kcal/mol for ,l structures). In helical structures cis arrangements of the peptide bond are only slightly less stable than their trans counterparts. This is no longer true for ,l structures, because the formation of an intramolecular hydrogen bond is possible only for trans peptide bonds. In most cases, proline and hydroxyproline show the same general trends; however, the electronegative 4(R) substituent of hydroxyproline leads to a strong preference for up puckerings irrespective of the backbone conformation. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 341,350, 2002 [source] Hydrolysis of the amyloid ,-peptide (A,) 1,40 between Asp23,Val24 produces non-aggregating fragments.JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2005An electrospray mass spectrometric study Abstract The aggregation of full-length (residues 1,40) amyloid ,-peptide (A,) and fragments corresponding to residues 1,23 and 24,40 was studied by electrospray mass spectrometry, using gramicidin as a non-aggregating reference. Following a lag period, A,(1,40) at 140 µM concentration aggregates with apparent first-order kinetics. Under acidic conditions A,(1,40) undergoes spontaneous cleavage between Asp23,Val24 and to a lesser extent also at two other Asp,X motifs. Incubation in acidic H218O showed incorporation of 18O in fragment A,(1,23), confirming that the Asp23,Val24 peptide bond had been hydrolyzed. Incubation of synthetic A,(1,23) and A,(24,40) peptides with A,(1,40) showed that A,(24,40) remained in solution for several months, that A,(1,23) partly disappeared from solution, whereas A,(1,40) completely disappeared. Further, treatment of sedimentable aggregates formed after co-incubation of the three peptides with hexafluoro-2-propanol or formic acid recovered the intensity of A,(1,40). These data support previous studies showing that the region of A, encompassing residues 16,24 is necessary for aggregation into amyloid fibrils. Copyright © 2005 John Wiley & Sons, Ltd. [source] Transition metal complexes of a cyclic pseudo hexapeptide: synthesis, complex formation and catalytic activities,,JOURNAL OF PEPTIDE SCIENCE, Issue 9 2008Huong Ngyen Abstract To contribute to a better understanding of metalloenzymes, we studied ion selectivity, complex formation tendencies and catalytic activities of linear and cyclic pseudopeptides. In this contribution, a linear and cyclic pseudo hexapeptide is described. The complex with transition metal ions and the sequence were designed using the programme COSMOS. Different routes of solid-phase synthesis were performed and compared using anchoring by C -terminus or a His side chain, using preformed pseudodipeptide building units or formation of N -functionalized peptide bond during stepwise assembly. The different strategies were compared regarding cyclization tendency, yield and purity. Side-chain anchoring to solid support favours the cyclization but leads to the formation of difficult to separate dioxopiperazine. Both routes require preformed building units. Complex-formation tendencies and selectivity for certain bivalent transition metal ions were experimentally estimated and compared to ones predicted theoretically. CD measurements indicate conformational changes by complex formation with different metal ions. Catalytic activities on oxidation of catechol and hydrolysis of bis-phosphate esters by some metal complexes of linear and cyclic peptide show only low catalytic activities compared to other model peptides and related metalloenzymes. The preference of the cyclic peptide for complexation of Ni2+ corresponds well to the predictions of COSMOS-NMR force field calculations. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Investigation of cis/trans ratios of peptide bonds in AVP analogues containing N -methylphenylalanine enantiomersJOURNAL OF PEPTIDE SCIENCE, Issue 1 2006Emilia Sikorska Abstract The solution conformation of vasopressin analogues, modified at positions 2 and 3 with N -methylphenylalanine or its enantiomer, [D -MePhe2,MePhe3]AVP and [MePhe2,D -MePhe3]AVP, were studied by 2D NMR spectroscopy in H2O/D2O and theoretical calculations (EDMC/ANALYZE). In the case of [MePhe2,D -MePhe3]AVP, the synthesis afforded two products, A and B, which had identical molecular ions and similar retention times on HPLC. This finding was explained by racemization of Cys1, which gave an additional analogue, [D -Cys1,MePhe2,D -MePhe3]AVP (B). The possibility is not excluded of racemization of Cys1 in the remaining analogues of this series. However, only in the case of [MePhe2,D -MePhe3]AVP was this process so distinct that two strong peaks in the HPLC chromatogram were observed. The NMR spectra of all the analogues showed several distinct sets of residual proton resonances. This suggests that the peptides adopt more than two groups of conformations in H2O/D2O. This fact is due to cis/trans isomerization. Two more populated isomers arise from the cis/trans isomerization across the 2,3 peptide bond in [D -MePhe2,MePhe3]AVP and [MePhe2,D -MePhe3]AVP (A) and across the 1,2 peptide bond in [D -Cys1,MePhe2,D -MePhe3]AVP (B). Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] New antitumour cyclic astin analogues: synthesis, conformation and bioactivityJOURNAL OF PEPTIDE SCIENCE, Issue 2 2004Dr Filomena Rossi Abstract Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical structures, consist of a 16-membered ring system containing a unique ,,,-dichlorinated proline [Pro(Cl)2], other non-coded amino acid residues and a cis conformation in one of the peptide bonds. The astin backbone conformation, along with the cis peptide bond in which the ,,,-dichlorinated proline residue is involved, was considered to play an important role in their antineoplastic activities on sarcoma 180A and P388 lymphocytic leukaemia in mice, but the scope and potential applications of this activity remain unclear. With the aim at improving our knowledge of the conformational properties influencing the bioactivity in this class of compounds, new astin-related cyclopeptides were synthesized differing from the natural products by the presence of some non-proteinogenic amino acid residues: Aib, Abu, -(S),3 -hPhe and a peptide bond surrogate (-SO2 -NH-). The analogues prepared c(-Pro-Thr-Aib-,3 -Phe-Abu-), c[Pro-Thr-Aib-(S),3 -hPhe-Abu], c[Pro-Abu-Ser-(S),3 -hPhe,(CH2 -SO2 -NH)-Abu] and c[Pro-Thr-Aib-(S),3 -hPhe,(CH2 -SO2 -NH)-Abu] were synthesized by classical methods in solution and tested for their antitumour effect. These molecules were studied by crystal-state x-ray diffraction analysis and/or solution NMR and MD techniques. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Mass spectrometric and chemical stability of the Asp-Pro bond in herpes simplex virus epitope peptides compared with X-Pro bonds of related sequencesJOURNAL OF PEPTIDE SCIENCE, Issue 8 2002Zsolt Skribanek Abstract The mass spectrometric analysis of the immunodominant epitope region (273,284) of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) showed a favoured fission at the Asp-Pro peptide bond. The fast atom bombardment collision induced dissociation (FAB-CID) study of closely related X-Pro peptides documented that neither the length nor the amino acid composition of the peptide has a significant influence on this preferential cleavage. At the same time the DP bond proved to be sensitive to acidic conditions in the course of peptide synthesis. These observations prompted us to compare the chemical and mass spectrometric stability of a new set of nonapeptides related to the 273,284 epitope region of gD, i.e. SALLEDPVG and SALLEXPVG peptides, where X = A, K, I, S, F, E or D, respectively. The chemical stability of these peptides during acidic hydrolysis was investigated by electrospray ionization mass spectrometry (ESI-MS) and the products were identified by ESI-MS and on-line high performance liquid chromatography,mass spectrometry (HPLC-MS). The mass spectrometric fragmentation and bond stability of the untreated peptide samples were also studied using ESI-MS and liquid secondary ion mass spectrometry (LSIMS). Both the chemical hydrolysis and the mass spectrometric fragmentation showed that the Asp-Pro bond could easily be cleaved, while the KP bond proved to be stable under both circumstances. On the other hand, the XP bond (X = A, I, S, F or E) fragmented easily under the mass spectrometric conditions, but was not sensitive to the acidolysis. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Influence of thermal motion on 1H chemical shifts in proteins: the case of bovine pancreatic trypsin inhibitorJOURNAL OF PEPTIDE SCIENCE, Issue 3 2001Bernard Busetta Abstract The possible influence of thermal motion on 1H chemical shifts is discussed for a small stable protein, the bovine pancreatic Kunitz trypsin inhibitor (BPTI). The thermal effects on the aromatic side chains and on the backbone are treated separately. The thermal motion of the aromatic side chains is accounted for in terms of their rotation around the C,C, bond and the motion of each individual proton is interpreted as a ratio between the amount of ordered and quite disordered states. The influence of hydrogen bonds is introduced as an extra contribution to the chemical shifts of the bonded proton. Their contribution to the chemical shifts resulting from the polarization of the peptide bond is investigated, as is their influence on local flexibility. Finally, the relative importance of each contribution to the chemical shift information is compared. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF-A2 domain as substrateJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2004J. L. Whitelock Summary., The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain. Measurement of ADAMTS-13 activity is a clinical utility for thrombotic diseases, but the current assays used for diagnostic and clinical research are non-physiological and time consuming. We have expressed in bacteria a recombinant VWF-A2 peptide (aa 718,905) that contains both a 6xHis tag at the N-terminal end and a Tag-100 epitope at the C-terminal end. Diluted plasma was mixed with the VWF-A2 peptide and digestion was allowed to proceed in a Ni2+ -coated microtiter well plate for 2 h. The immobilized Ni2+ captures the VWF-A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C-terminus fragment of the A2 peptide that contains the Tag-100. The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag-100 epitope by the antiTag-100 monoclonal antibody. The assay was completed in <5 h. We then used the assay to analyze ADAMTS-13 activity in plasma from 39 healthy donors and 16 samples from patients diagnosed as thrombotic thrombocytopenic purpura. The average of enzyme activity ±,SEM for normal plasmas diluted 1 : 50 was 40 ± 4.2% while the value obtained for the patients was 2.4 ± 0.7%. These results were validated by a traditional long incubation assay (24 h). Our assay provides significant advantages over currently used assays because it is quicker, reproducible, cost effective and measures ADAMTS-13 activity under physiological and non-denaturing conditions. This assay is clinically useful and significant in measuring ADAMTS-13 activity in plasma. [source] Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitorsPROTEIN SCIENCE, Issue 3 2010Susmita Khamrui Abstract For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed "mini-proteins," containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECIL -WCIS, ETIL -WCIS, and STIL -WCIS, where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECIL -WCIS and STIL -WCIS behave like good inhibitors, ETIL -WCIS behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETIL -WCIS shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETIL -WCIS leading to a loss of canonical conformation, explaining its substrate-like behavior. Incorporation of this barrier back in ETIL -WCIS through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop-scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design. [source] Alteration of the disulfide-coupled folding pathway of BPTI by circular permutationPROTEIN SCIENCE, Issue 5 2004Grzegorz Bulaj BPTI, bovine pancreatic trypsin inhibitor; cBPTI, a circular form of BPTI generated by forming a peptide bond between the natural termini; cpBPTI, circularly permuted BPTI. Abstract The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3,8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the ,-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein. [source] Modeling of the bacterial luciferase-flavin mononucleotide complex combining flexible docking with structure-activity dataPROTEIN SCIENCE, Issue 8 2001Leo Yen-Cheng Lin FMN, flavin mononucleotide; FMNH2, reduced FMN Abstract Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown. The determined location of the phosphate-binding site close to Arg 107 on the , subunit of luciferase is supported here by point mutagenesis. This information, together with previous structure-activity data for the length of the linker connecting the phosphate group to the isoalloxazine ring represent important characteristics of the luciferase-bound conformation of the flavin mononucleotide. A model of the luciferase,flavin complex is developed here using flexible docking supplemented by these structural constraints. The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation search by using the Monte Carlo minimization approach. The resulting databases of energy-ranked feasible conformations of the luciferase complexes with flavin mononucleotide, ,-phosphopentylflavin, ,-phosphobutylflavin, and ,-phosphopropylflavin were filtered according to the structure-activity profile of these analogs. A unique model was sought not only on energetic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the luciferase-binding site, an orientation that is also inaccessible to the inactive flavin analog. The resulting model of the bacterial luciferase,flavin mononucleotide complex is consistent with the experimental data available in the literature. Specifically, the isoalloxazine ring of the flavin mononucleotide interacts with the Ala 74,Ala 75 cis -peptide bond as well as with the Cys 106 side chain in the , subunit of luciferase. The model of the binary complex reveals a distinct cavity suitable for aldehyde binding adjacent to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site. [source] Electrospray tandem mass spectrometry of lexitropsinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2001M. Rosário M. Domingues Several compounds, representative of the class of lexitropsins, were analyzed by electrospray tandem mass spectrometry. The study of the fragmentations of the protonated molecular species ([M,+,H]+) and of selected fragment ions allowed proposals for the main fragmentation pathways of compounds of this type. The interpretation of the fragmentation pathways of these compounds was complicated because of intramolecular hydrogen migration. In order to better understand the fragmentation pathways, the MS/MS/MS spectra of several compounds, and the MS/MS and MS/MS/MS spectra of the deuterated compounds, were obtained. Accurate mass measurements helped elucidate the structures of smaller fragment ions. Low-energy collision-induced decomposition (CID) tandem mass spectrometry of lexitropsins with electrospray ionization has proven to be a good method for the structural characterization and identification of this class of compounds. Main fragmentation pathways occur by cleavage of the peptide bond followed by the elimination of the substituted pyrrole ring, and their elucidation will facilitate structural characterization of new lexitropsins. Copyright © 2001 John Wiley & Sons, Ltd. [source] Crystallization and preliminary X-ray crystallographic analysis of blood coagulation factor V-activating proteinase (RVV-V) from Russell's viper venomACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Daisuke Nakayama Russell's viper venom blood coagulation factor V activator (RVV-V) is a thrombin-like serine proteinase that specifically activates factor V by cleaving a single peptide bond between Arg1545 and Ser1546. Activated factor V combines with activated factor X produced by the enzyme RVV-X in the venom to form the prothombinase complex, which can induce disseminated intravascular coagulopathy in envenomated animals. In the current study, RVV-V was crystallized in order to attempt to understand its substrate specificity for factor V. Four distinct crystal forms of RVV-V were obtained using the sitting-drop vapour-diffusion method and diffraction data sets were collected on SPring-8 beamlines. The best crystal of RVV-V generated data sets to 1.9,Ĺ resolution. [source] Ribosomal crystallography: Peptide bond formation and its inhibitionBIOPOLYMERS, Issue 1 2003Anat Bashan Abstract Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3, end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination. © 2003 Wiley Periodicals, Inc. Biopolymers, 2003 [source] Expression of protein gene product 9·5 (PGP9·5)/ubiquitin-C-terminal hydrolase 1 (UCHL-1) in human myeloma cellsBRITISH JOURNAL OF HAEMATOLOGY, Issue 3 2004T. Otsuki Summary The neuron cytoplasmic protein gene product 9·5 (PGP9·5)/ubiquitin-C-terminal hydrolase 1 (UCHL-1) protein is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal of ubiquitin, and is involved in the processing of ubiquitin precursors and ubiquinated proteins. Although this molecule is known as a specific tissue marker for the neuroendocrine system, many reports have indicated that PGP9·5 is a marker for certain tumour types, such as cancer of the lung, colon, and pancreas. The expression of PGP9·5 in myeloma cells was examined. PGP9·5 seemed to be expressed specifically in myeloma cells as compared with other haematological malignant cells. In addition, in myeloma cells subjected to growth-factor starvation, the upregulation of PGP9·5 was observed in association with that of p27Kip1, a cyclin-dependent-kinase inhibitor, although the upregulation caused by irradiation was milder. In contrast, the hypoxic culture of myeloma cells induced down-regulation of PGP9·5. These results suggested that PGP9·5 may be a good marker for myeloma among haematological malignancies. In addition, it may indicate certain cellular features of myeloma cells, such as sensitivity to proteasome inhibitors. [source] Lasioglossins: Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae)CHEMBIOCHEM, Issue 12 2009Václav, ovský Dr. Abstract Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH2 (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH2 (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH2 (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of ,-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved ,-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH2 to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification. [source] From pro defensins to defensins: synthesis and characterization of human neutrophil pro ,-defensin-1 and its mature domainCHEMICAL BIOLOGY & DRUG DESIGN, Issue 2 2003Z. Wu Abstract: Human neutrophil ,-defensins (HNPs) are small, cationic, Cys-rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre-proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro-peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45-residue pro-peptide and the C-terminal functional domain. Here we described, total chemical synthesis of the 75-residue human neutrophil pro ,-defensin-1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met45,Ala46 peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys1,Cys6, Cys2,Cys4 and Cys3,Cys5, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro-peptide binds to HNP1 intermolecularly with an apparent Kd value of 6.2 ,m at pH 7.4, confirming the mode of intramolecular inactivation of human ,-defensin precursors. [source] The Proteolytic Stability of ,Designed' , -Peptides Containing , -Peptide-Bond Mimics and of Mixed ,,, -Peptides: Application to the Construction of MHC-Binding PeptidesCHEMISTRY & BIODIVERSITY, Issue 5 2005David Whereas , -peptides are rapidly degraded in vivo and in vitro by a multitude of peptidases, substrates constructed entirely of or incorporating homologated , -amino acid (i.e., , -amino acid) units exhibit a superior stability profile. Efforts made so far to proteolytically hydrolyze a ,, peptide bond have not proved fruitful; a study aimed at breaching this proteolytic stability is discussed here. A series of such bonds have been designed with side-chain groups similar in relative positions (constitution) and three-dimensional arrangements (configuration) as found about , -peptidic amide bonds. Increasing the prospect for degradation would permit the tuning of , -peptide stability; here, however, no cleavage was observed (1, 2, 4,6, Table,1). Peptides comprised of , - and , -amino acids (mixed ,,, -peptides, 8,11) are expected to benefit from both recognition by a natural receptor and a high level of proteolytic stability, ideal characteristics of pharmacologically active compounds. ,3 -Peptides containing , -amino acid moieties at the N-terminus are degraded, albeit slowly, by several peptidases. Of particular interest is the ability of pronase to cleave an ,, peptide bond, namely that of ,Ala,3hAla. Significantly, successful hydrolysis is independent of the configuration of the , -amino acid. Some of the ,,, -peptides discussed here are being investigated for their binding affinities to class I MHC proteins. The computer-programming steps required to prepare ,,, -peptides on an automated peptide synthesizer are presented. [source] The Trifluoroethylamine Function as Peptide Bond ReplacementCHEMMEDCHEM, Issue 12 2007Monica Sani Dr. 3D instead of 2D. That's the main difference between the tetrahedral stereogenic trifluoroethylamine function and the planar peptide bond. This property, together with other remarkable features such as high metabolic stability and low basicity, turns out to be of great importance to allow trifluoroethylamines to be excellent peptide bond replacements. [source] NUB1-mediated targeting of the ubiquitin precursor UbC1 for its C-terminal hydrolysisFEBS JOURNAL, Issue 5 2004Tomoaki Tanaka NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. Previously, we identified a negative regulator of the NEDD8 conjugation system, NEDD8 ultimate buster-1 (NUB1), that recruits NEDD8 and its conjugates to the proteasome for degradation. Recently, we performed yeast two-hybrid screening with NUB1 as bait and isolated a ubiquitin precursor UbC1 that is composed of nine tandem repeats of a ubiquitin unit through ,-peptide bonds. Interestingly, NUB1 interacted with UbC1 through its UBA domain. Further study revealed that the UBA domain interacted with ,-peptide bond-linked polyubiquitin, but not with isopeptide bond-linked polyubiquitin, indicating that the UBA domain of NUB1 is a specific acceptor for the linear ubiquitin precursor. A functional study revealed that an unidentified protein that was immunoprecipitated with NUB1 served as a ubiquitin C-terminal hydrolase for UbC1. Thus, NUB1 seems to form a protein complex with the unidentified ubiquitin C-terminal hydrolase and recruit UbC1 to this complex. This might allow the ubiquitin C-terminal hydrolase to hydrolyze UbC1, in order to generate ubiquitin monomers. Northern blot analysis showed that the mRNAs of both NUB1 and UbC1 were enriched in the testis. Furthermore, in situ hybridization showed that both mRNAs were strongly expressed in seminiferous tubules of the testis. These results may imply that the UbC1 hydrolysis mediated by NUB1 is involved in cellular functions in the seminiferous tubules such as spermatogenesis. [source] Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetasesFEMS MICROBIOLOGY LETTERS, Issue 1 2005Niran Roongsawang Abstract Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are l -peptidyl donors, d -peptidyl donors, and N -acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from l to d -form of incorporating amino acid acceptor occurs during or after peptide bond formation. l -peptidyl donors and d -peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process. [source] A Novel Synthesis of Highly Substituted Perhydropyrrolizines, Perhydroindolizines, and Pyrrolidines: Inhibition of the Peptidyl-Prolyl cis/trans Isomerase (PPIase) Pin1HELVETICA CHIMICA ACTA, Issue 2 2007Romain Siegrist Abstract In this paper, we describe the synthesis and biological evaluation of highly substituted perhydropyrrolizines that inhibit the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1, an oncogenic target. The enzyme selectively catalyzes the cis/trans isomerization of peptide bonds between a phosphorylated serine or threonine, and proline, thereby inducing a conformational change. Such structural modifications play an important role in many cellular events, such as cell-cycle progression, transcriptional regulation, RNA processing, as well as cell proliferation and differentiation. Based on computer modeling (Fig.,2), the new perhydropyrrolizinone derivatives (,)- 1a,b, decorated with two substituents, were selected and synthesized (Schemes,1,3). While enzymatic assays showed no biological activity, 15N,1H-HSQC-NMR spectroscopy revealed that (,)- 1a,b bind to the WW recognition domain of Pin1, apparently in a mode that does not inhibit PPIase activity. To enforce complexation into the larger active site rather than into the tighter WW domain of Pin1 and to enhance the overall binding affinity, we designed a perhydropyrrolizine scaffold substituted with additional aromatic residues (Fig.,5). A novel, straightforward synthesis towards this class of compounds was developed (Schemes,4 and 5), and the racemic compounds (±)- 22a,22d were found to inhibit Pin1 with Ki values (Ki,=,inhibition constant) in the micromolar range (Table,2). To further enhance the potency of these inhibitors, the optically pure ligands (+)- 22a and (+)- 33b,c were prepared (Schemes,6 and 7) and shown to inhibit Pin1 with Ki values down to the single-digit micromolar range. According to 15N,1H-HSQC-NMR spectroscopy and enzymatic activity assays, binding occurs at both the WW domain and the active site of Pin1. Furthermore, the new synthetic protocol towards perhydropyrrolizines was extended to the preparation of highly substituted perhydroindolizine ((±)- 43; Scheme,8) and pyrrolidine ((±)- 48a,b; Scheme,9) derivatives, illustrating a new, potentially general access to these highly substituted heterocycles. [source] A modified Ising model for the thermodynamic properties of local and global protein folding,unfolding observed by circular dichroism and small-angle X-ray scatteringJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2007Ying-Jen Shiu Based on the mean-field approximation, we have applied a modified Ising model to describe general protein unfolding behavior at thermodynamic equilibrium with the free energy contributed by the subgroup units (amino acids or peptide bonds) of the protein. With the thermodynamic properties of the protein, this model can associate the stepwise change of an unfolding fraction ratio profile with the local and global conformation unfolding. Taking cytochrome c (cyt c) as a model protein, we have observed, using small-angle X-ray scattering and circular dichroism (CD), the global and local structure changes for the protein in three kinds of denaturant environments: acid, urea and guanidine hydrochloride. The small-angle X-ray scattering and CD results are mapped to the unfolding fractions as a function of the pH value or denaturant concentration, from which we have extracted local and global unfolding free energies of cyt c in different denaturant environments using a modified Ising model. Based on the characteristics of the thermodynamic properties deduced from the local and global protein folding,unfolding, we discuss the thermodynamic stabilities of the protein in the three denaturant environments, and the possible correlation between the global conformation change of the protein and the local unfolding activities of the S,Fe bond in the Met80-heme and the ,-helices. [source] Quantum mechanical study of the conformational behavior of proline and 4R-hydroxyproline dipeptide analogues in vacuum and in aqueous solutionJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 3 2002Caterina Benzi The conformational behavior of the title compounds has been investigated by Hartree,Fock, MP2, and DFT computations on the most significant structures related to variations of the backbone dihedral angles, cis/trans isomerism around the peptide bond, and diastereoisomeric puckering of the pyrrolidine ring. In vacuum the reversed , turn (,l), characterized by an intramolecular hydrogen bridge, corresponds to the absolute energy minimum for both puckerings (up and down) of the pyrrolidine ring. An additional energy minimum is found in the helix region, but only for an up puckering of the pyrrolidine ring. When solvent effects are included by means of the polarizable continuum model the conformer observed experimentally in condensed phases becomes the absolute minimum. The down puckering is always favored over its up counterpart, albeit by different amounts (0.4,0.5 kcal/mol for helical structures and about 2 kcal/mol for ,l structures). In helical structures cis arrangements of the peptide bond are only slightly less stable than their trans counterparts. This is no longer true for ,l structures, because the formation of an intramolecular hydrogen bond is possible only for trans peptide bonds. In most cases, proline and hydroxyproline show the same general trends; however, the electronegative 4(R) substituent of hydroxyproline leads to a strong preference for up puckerings irrespective of the backbone conformation. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 341,350, 2002 [source] Conformational analysis of thiopeptides: derivation of sp2 sulfur parameters for the CFF91 force fieldJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 10 2001Tran Trung Tran Abstract When a sulfur atom is used to substitute for the oxygen in peptide bonds, its bulkiness should restrict the conformational space available to an amino acid. This conformational restriction as well as the ability to confer resistance to enzymatic degradation in the body means that thio-substituted amino acids are potentially useful building blocks for drug design. To simulate the effects of thio substitution, force field parameters for sp2 sulfur are required. In this article, parameters for the thioamide group have been derived for the molecular mechanics CFF91 force field (available at http://www.ludwig.edu.au/archive/tran). The bond increment charges were obtained by fitting to ab initio charges and dipoles. The van der Waals parameters were obtained by fitting to high-resolution crystallographic data, and the nonbonded parameters were verified by comparing with experimentally derived lattice energy. The bonded parameters were derived by least-square fits to the ab initio calculated energy surfaces, i.e., conformational energy as well as their first and second derivatives of seven model thioamide molecules. When the sp2 sulfur parameters were tested on a set of seven X-ray crystallographic structures from the Cambridge Structural Database, they satisfactorily reproduced the bond lengths, bond angles, torsional angles, and nonbonded distances of all the crystal structures. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1010,1025, 2001 [source] PROTEINASES IN HYBRID CATFISH VISCERA: CHARACTERIZATION AND EFFECT OF EXTRACTION MEDIAJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT Proteolytic activity from viscera extract of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was investigated. Optimal pH and temperature for casein hydrolysis were 9.0 and 50C, respectively. The enzyme was stable to heat treatment up to 40C and over a pH range of 7,11 for 30,120 min. The proteolytic activity was effectively inhibited by soybean trypsin inhibitor, benzamidine, phenylmethylsulfonyl fluoride and N -p-tosyl-L-lysine chloromethyl ketone. Activities of the viscera extract continuously decreased as NaCl concentration increased, while activities increased as CaCl2 concentration increased. Based on the proteinase activity of zones separated by electrophoresis, the molecular mass of the major proteinases in hybrid catfish viscera was 23 and 20 kDa. The effect of extraction media on recovery of proteinases was also studied. Extraction of the viscera powder with 50 mM Tris-HCl, pH 7.0 containing 0.5 M NaCl and 0.2% (v/v) Brij 35 rendered a higher recovery of proteinase activity than other extractants tested (P < 0.05). The results suggested that major proteinases in hybrid catfish viscera were heat-activated alkaline proteinases, most likely trypsin-like serine proteinases. PRACTICAL APPLICATIONS Hybrid catfish viscera is an abundant and underutilized resource that can be used as a unique proteinase source. Proteinase from various sources catalyzes the hydrolysis of peptide bonds. Thus, it is expected that like other proteinases, hybrid catfish proteinase would be useful in biomedical, food and beverage application. Moreover, the presented extraction media could be adopted to recover the trypsin-like serine proteinase from hybrid catfish viscera, which is currently a solid waste of Pa-duk-ra industry. 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