RSSEffects of Charge Location on the Absorption by and Lifetimes of Protonated Tyrosine Peptides in Vacuo
Abstract
Nearby charges affect the electronic energy levels of chromophores, with the extent of the effect being determined by the magnitude of the charge and degree of charge-chromophore separation. The molecular configuration dictates the charge-chromophore distance. Hence, in this study we aim to assess how the location of the charge influences the absorption of a set of model protonated and diprotonated peptide ions, and whether spectral differences are large enough to be identified. The studied ions were the dipeptide YK, the tripeptide KYK (Y = tyrosine, K = lysine) and their complexes with 18-crown-6-ether (CE). The CE targets the ammonium group by forming internal ionic hydrogen bondsand limits the folding of the peptide. In the tripeptide, the distance between the chromophore and the backbone ammonium is enlarged relative to that in the dipeptide. Experiments were performed in an electrostatic ion storage ring using a tunable laser system, and action spectra based on lifetime measurements were obtained in the range from 210 nm to 310 nm. The spectra are all quite similar though there seems to be some changes in the absorption band between 210 nm and 250 nm, while in the lower energy band all ions had a maximum absorption at ~275 nm. Lifetimes after photoexcitation were found to shorten upon protonation and lengthen upon CE complexation, in accordance with the increased number of degrees of freedom and an increase in activation energies for dissociation as the mobile proton model is no longer operative.
Perturbing the oxidizing environment of the periplasm stimulates the PhoQ/PhoP system in E. coli.
Source
Department of Biology, University of Pennsylvania, 433 S. University Ave Philadelphia, PA 19104-6018.
Abstract
The PhoQ/PhoP two-component system is repressed by divalent cations such as Mg(2+) and Ca(2+) in the growth medium and stimulated by low pH, and certain cationic antimicrobial peptides. In E. coli, it has recently been shown that the histidine kinase PhoQ is also modulated by at least two additional factors, the small membrane proteins SafA and MgrB. This raises the possibility that the PhoQ/PhoP circuit has additional regulatory components and integrates additional input signals. We screened E. coli transposon insertion mutants to look for proteins that modulate the activity of the PhoQ/PhoP system and uncovered a role for DsbA, a periplasmic oxidant that facilitates the formation of disulfidebonds. Deletion of dsbA or dsbB, which maintains a pool of oxidized DsbA, leads to increased transcription of at least two PhoP-regulated genes. Addition of the reducing agent DTT to wild-type cells has a similar effect and treatment of a dsbA null strain with the oxidant Cu(2+)rescues the reporter gene expression phenotype. We also demonstrate that expression of an MgrB mutant that lacks cysteines blocks the effect of a dsbA null mutation on PhoQ/PhoP activity, suggesting that MgrB acts downstream of DsbA in this pathway. Taken together, these results demonstrate that a decrease in the oxidizing activity of the periplasm stimulates PhoQ/PhoP and may reveal a new input stimulus for this important two-component system.
Characterization of Pt-protein complexes by nHPLC-ESI-LTQ MS/MS using a gel-based bottom-up approach.
Source
Department of Analytical Chemistry, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain.
Abstract
The suitability of in-gel digestion for the characterization of Pt-binding proteins by gel-based bottom-up MS approaches has been evaluated regarding the preservation of Pt-protein bonds during the process. Standard proteins (albumin, transferrin, carbonic anhydrase, myoglobin and cytochome c) incubated with cisplatin were separated by nrSDS-PAGE and in-gel trypsin-digested. The whole in-gel digestion protocol included treatment with reagents such as: ammonium bicarbonate, acetonitrile, formic acid, trypsin as enzyme and alternatively, dithiotreitol and iodoacetamide as reducing and alkylating agents. Digests were analyzed by nHPLC-ESI-LTQ-MS/MS and Pt-peptides were recognized in all the proteins studied on the basis of their isotopic pattern. Only when the reducing and alkylating reagents were used, the amount of detectable Pt-peptides decreased due to the high reactivity of thiol containing reagents towards Pt. Furthermore, the repeated use of acetonitrile could lead to the replacement of ligands originally attached to Pt by CN(-), but does not affect the Pt-protein binding. Platinum-binding sites on the proteins were elucidated from the CID-MS/MS fragmentation spectra and assessed by evaluation of protein structures. Several histidines, cysteines and methionines were identified as platinum binding sites in the different standard proteins. Results were in accordance to those obtained with in-solution digestions.
Copyright © 2011 Elsevier B.V. All rights reserved.
Primordial soup or vinaigrette: did the RNA world evolve at acidic pH?
Abstract
ABSTRACT:
BACKGROUND:
The RNA world concept has wide, though certainly not unanimous, support within the origin-of-life scientific community. One view is that life may have emerged as early as the Hadean Eon 4.3-3.8 billion years ago with an atmosphere of high CO2 producing an acidic ocean of the order of pH 3.5-6. Compatible with this scenario is the intriguing proposal that life arose near alkaline (pH 9-11) deep-sea hydrothermal vents like those of the 'Lost City', with the interface with the acidic ocean creating a proton gradient sufficient to drive the first metabolism. However, RNA is most stable at pH 4-5 and is unstable at alkaline pH, raising the possibility that RNA may have first arisen in the acidic ocean itself (possibly near an acidic hydrothermal vent), acidic volcanic lake or comet pond. As the Hadean Eon progressed, the ocean pH is inferred to have gradually risen to near neutral as atmospheric CO2 levels decreased.
PRESENTATION OF THE HYPOTHESIS:
We propose that RNA is well suited for a world evolving at acidic pH. This is supported by the enhanced stability at acidic pH of not only the RNA phosphodiester bond but also of the aminoacyl-(t)RNA and peptide bonds. Examples of in vitro-selected ribozymes with activities at acid pH have recently been documented. The subsequent transition to a DNA genome could have been partly driven by the gradual rise in ocean pH, since DNA has greater stability than RNA at alkaline pH, but not at acidic pH.
TESTING THE HYPOTHESIS:
We have proposed mechanisms for two key RNA world activities that are compatible with an acidic milieu: (i) non-enzymatic RNA replication of a hemi-protonated cytosine-rich oligonucleotide, and (ii) specific aminoacylation of tRNA/hairpins through triple helix interactions between the helical aminoacyl stem and a single-stranded aminoacylating ribozyme.
IMPLICATIONS OF THE HYPOTHESIS:
Our hypothesis casts doubt on the hypothesis that RNA evolved in the vicinity of alkaline hydrothermal vents. The ability of RNA to form protonated base pairs and triples at acidic pH suggests that standard base pairing may not have been a dominant requirement of the early RNA world. Reviewers This article was reviewed by Eugene Koonin, Anthony Poole and Charles Carter (nominated by David Ardell).
The desoxazoline asidiacyclamide analogue cyclo(Gly-Thr-D-Val-Thz-Ile-Thr-D-Val-Thz) acetonitrile monosolvate.
Source
Osaka University of Pharmaceutical Sciences, Nasahara, Osaka 569-1094, Japan.
Abstract
THE TITLE PEPTIDE [SYSTEMATIC NAME: 4-(butan-2-yl)-7,20-bis-(1-hy-droxy-eth-yl)-10,23-bis-(propan-2-yl)-12,25-dithia-3,6,9,16,19,22,27,28-octa-aza-tricyclo-[22.2.1.1(11,14)]octa-cosa-1(26),11(28),13,24(27)-tetra-ene-2,5,8,15,18,21-hexone acetonitrile monosolvate], C(32)H(48)N(8)O(8)S(2)·CH(3)CN, an analogue of ascidiacyclamide (ASC) [cyclo(-Ile-Oxz-D-Val-Thz-)(2)], lies about a twofold rotation axis, so that the glycine (Gly) and isoleucine (Ile) residues are each disordered over two sites with equal occupancies. The acetonitrile mol-ecule is also located on a twofold axis passing through the C and N atoms. In the peptide, the thia-zole rings are faced to each other with a dihedral angle of 9.63 (15)° and intra-molecular N-H⋯O and O-H⋯O hydrogen bonds are observed. A bifurcated N-H⋯(O,O) hydrogen bond links thepeptide mol-ecules into a layer parallel to the ab plane.
Cyclo-linopeptide B methanol tris-olvate.
Abstract
The title compound, C(56)H(83)N(9)O(9)S·3CH(3)OH, is a methanol tris-olvate of the cyclo-linopeptide cyclo(Met(1)-Leu(2)-Ile(3)-Pro(4)-Pro(5)-Phe(6)-Phe(7)-Val(8)-Ile(9)) (henceforth referred to as CLP-B), which was isolated from flaxseed oil. All the amino acid residues are in an l-configuration based on the CORN rule. The cyclic nona-peptideexhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α-turn and two consecutive β-turns each containing a N-H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) or the third residue (β-turns), repectively. In the crystal, the components of the structure are linked by N-H⋯O and O-H⋯O hydrogen bondsinto chains parallel to the a axis.
The Serine-Proline Turn: A Novel Hydrogen-Bonded Template for Designing Peptidomimetics.
Source
Center for Advanced Drug Research (CADRE), SRI International, 140 Research Drive, Harrisonburg, Virginia 22802, United States.
Abstract
Serine-Proline (SP) dipeptide motifs have been shown to form unique hydrogen-bonding patterns in protein crystal structures. Peptides were designed to mimic these patterns by forming the 6 + 10 and the 9 + 10 hydrogen-bonded rings. Factors that contribute to the formation of SP turns include controlling backbone flexibility and amino acid chirality along with creating a hydrophobic environment around the intramolecular hydrogen bonds.
Demonstration of α-Helical Structure of Peptides Tethered to Gold Surfaces Using Surface Infrared and Circular Dichroic Spectroscopies.
Abstract
Gold and quartz surfaces terminated in an alkane thiol self-assembled monolayer (SAM) that were partially terminated with azide were reacted with a helical peptide containing two alkyne groups in a Cu(I)-catalyzed Huisgen cycloaddition. Surface grazing incidence angle reflection-absorption infrared spectroscopy (GRAS-IR) was used to determine that when the Au surface was terminated with 25% of the monolayer containing azide groups, 92% of available azide groups reacted with the peptide. The majority of peptides reacted with both alkynes, resulting in peptides tethered to the surface through two covalent bonds. This was confirmed by comparison to a control peptide containing only one reactive alkyne group. Surface circular dichroic (CD) spectroscopy showed that while the helical structure of the peptide was distorted in the reaction solution, α-helical structure was induced when tethered on the SAM functionalized Au surface. Demonstration of the preservation of desired secondary structure of helical elements at a chemically functionalized surface is an important advance in preparing robust biologically mimetic surfaces to integrate functioning proteins into inorganic materials.
Role of amino acid hydrophobicity, aromaticity, and molecular volume on IAPP(20-29) amyloid self-assembly.
Source
Department of Chemistry, University of Rochester, Rochester, New York 14627.
Abstract
Aromatic amino acids strongly promote cross-β amyloid formation; whether the amyloidogenicity of aromatic residues is due to high hydrophobicity and β-sheet propensity or formation of stabilizing π-π interactions has been debated. To clarify the role of aromatic residues on amyloid formation, the islet amyloid polypeptide 20-29 fragment [IAPP(20-29)], which contains a single aromatic residue (Phe 23), was adopted as a model. The side chain of residue 23 does not self-associate in cross-β fibrils of IAPP(20-29) (Nielsen et al., Angew Chem Int Ed 2009;48:2118-2121), allowing investigation of the amyloidogenicity of aromatic amino acids in a context where direct π-π interactions do not occur. We prepared variants of IAPP(20-29) in which Tyr, Leu, Phe, pentafluorophenylalanine (F5-Phe), Trp, cyclohexylalanine (Cha), α-naphthylalanine (1-Nap), or β-naphthylalanine (2-Nap) (in order of increasing peptide hydrophobicity) were incorporated at position 23 (SNNXGAILSS-NH2), and the kinetic and thermodynamic effects of these mutations on cross-β self-assembly were assessed. The Tyr, Leu, and Trp 23 variants failed to readily self-assemble at concentrations up to 1.5 mM, while the Cha 23 mutant fibrillized with attenuated kinetics and similar thermodynamic stability relative to the wild-type Phe 23 peptide. Conversely, the F5-Phe, 1-Nap, and 2-Nap 23 variants self-assembled at enhanced rates, forming fibrils with greater thermodynamic stability than the wild-type peptide. These results indicate that the high amyloidogenicity of aromatic amino acids is a function of hydrophobicity, β-sheet propensity, and planar geometry and not the ability to form stabilizing or directing π-π bonds. Proteins 2012;. © 2011 Wiley Periodicals, Inc.
Copyright © 2011 Wiley Periodicals, Inc.
Chain length effects on helix-hairpin distribution in short peptides with Aib-(D) Ala and Aib-Aib Segments.
Source
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Abstract
The Aib-(D) Ala dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-(D) Ala-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7), and Boc-Aib-Xxx-NHMe (4, 8), where Xxx = (D) Ala, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7), and Boc-Aib-Aib-NHMe (8) helical conformations have been established by NMR studies in both hydrogen bonding (CD(3) OH) and non-hydrogen bonding (CDCl(3) ) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-(D) Ala-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl(3) and β-hairpin conformations in CD(3) OH. The β-turn conformations (type-I'/III) stabilized by intramolecular 4→1 hydrogen bonds are observed for the peptide Boc-Aib-(D) Ala-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 3(10) -helical conformation stabilized by three 4→1 hydrogen bonds. The peptide Boc-Val-Aib-(D) Ala-Leu-NHMe (3) adopts a novel α-turn conformation, stabilized by three intramolecular hydrogen bonds (two 4→1 and one 5→1). The Aib-(D) Ala segment adopts a type-I' β-turn conformation. The observation of an NOE between Val (1) NH↔HNCH(3) (5) in CD(3) OH suggests, that the solid state conformation is maintained in methanol solutions. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 744-756, 2011.
Copyright © 2011 Wiley Periodicals, Inc.
Quantitative protease cleavage site profiling using Tandem-Mass-Tag labeling and LC-MALDI-TOF/TOF MS/MS analysis.
Abstract
Knowledge of cleavage site specificity and activity are major prerequisites for understanding protease function. Based on a recently presented approach for proteomic identification of cleavage sites (PICS) in proteome-derived peptide libraries, we developed an isobaric labeling quantitative LC-MALDI-TOF/TOF MS/MS approach (Q-PICS) for simultaneous determination of cleavage site specificity and robust relative quantification of proteolytic events. For GluC-protease, 737 cleavage sites were identified in a yeast proteome-derived peptide library; 94.0% showed the typical GluC specificity forpeptide bonds at glutamyl and aspartyl residues. The six-plex tandem mass tagging strategy allowed for three simultaneous replicates in a single run, guaranteeing high confidence and robust statistics for quantitative measurements. Using the quantitative capacity of Q-PICS, we performed a comparison of cleavage site specificity of GluC in two different buffer systems. The results support earlier findings describing that apparent difference between the buffer systems are probably caused by the inhibitory effect of bicarbonate on the overall GluC activity, and that the preference for Glu-X bonds compared to Asp-X bonds is independent of the buffer system used.
Mass spectrometry-based proteomics strategies for protease cleavage site identification.
Source
AG Systematische Proteomforschung, Institut für Experimentelle Medizin, Christian-Albrechts-Universität, Kiel, Germany. b.vdberg@iem.uni-kiel.de.
Abstract
Protease-catalyzed hydrolysis of peptide bonds is one of the most pivotal post-translational modifications fulfilling manifold functions in the regulation of cellular processes. Therefore, dysregulation of proteolytic reactions plays a central role in many pathophysiological events. For this reason, understanding the molecular mechanisms in proteolytic reactions, in particular the knowledge of proteases involved in complex processes, expression levels and activity of protease and knowledge of the targeted substrates are an indispensable prerequisite for targeted drug development. The present review focuses on mass spectrometry-based proteomic methods for the analysis of protease cleavage sites, including the identification of the hydrolyzed bonds as well as of the surrounding sequence. Peptide- and protein-centric approaches and bioinformatic tools for experimental data interpretation will be presented and the major advantages and drawbacks of the different approaches will be addressed. The recent applications of these approaches for the analysis of biological function of different protease classes and potential future directions will be discussed.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
A Cyclic Peptide Inhibitor of ApoC-II Peptide Fibril Formation: Mechanistic Insight from NMR and Molecular Dynamics Analysis.
Source
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.
Abstract
The misfolding and aggregation of proteins to form amyloid fibrils is a characteristic feature of several common age-related diseases. Agents that directly inhibit formation of amyloid fibrils represent one approach to combating these diseases. We have investigated the potential of a cyclic peptide to inhibit fibril formation by fibrillogenic peptides from human apolipoprotein C-II (apoC-II). Cyc[60-70] was formed by disulfide cross-linking of cysteine residues added to the termini of the fibrillogenic peptide comprising apoC-II residues 60-70. This cyclic peptide did not self-associate into fibrils. However, substoichiometric concentrations of cyc[60-70] significantly delayed fibril formation by the fibrillogenic, linear peptides apoC-II[60-70] and apoC-II[56-76]. Reduction of the disulfide bond or scrambling the amino acid sequence within cyc[60-70] significantly impaired its inhibitory activity. The solution structure of cyc[60-70] was solved using NMR spectroscopy, revealing a well-defined structure comprising a hydrophilic face and a more hydrophobic face containing the Met60, Tyr63, Ile66 and Phe67 side chains. Molecular dynamics (MD) studies identified a flexible central region within cyc[60-70], while MD simulations of "scrambled" cyc[60-70] indicated an increased formation of intramolecular hydrogen bonds and a reduction in the overall flexibility of the peptide. Our structural studies suggest that the inhibitory activity of cyc[60-70] is mediated by an elongated structure with inherent flexibility and distinct hydrophobic and hydrophilic faces, enabling cyc[60-70] to interact transiently with fibrillogenic peptides and inhibit fibril assembly. These results suggest that cyclic peptides based on amyloidogenic core peptides could be useful as specific inhibitors of amyloid fibril formation.
Copyright © 2011. Published by Elsevier Ltd.
Photodissociation pathways and lifetimes of protonated peptides and their dimers.
Source
Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus C, DenmarkChemistry Department, Moscow State University, Moscow 119991, Russia and Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus C, DenmarkUniversité de Lyon, F-69622, Lyon, France and Université Lyon 1, Villeurbanne, CNRS, UMR 5579, LASIM, FranceUniversité de Lyon, F-69622, Lyon, France and Université Lyon 1, Villeurbanne, CNRS, UMR 5180, Sciences Analytiques, France.
Abstract
Photodissociation lifetimes and fragment channels of gas-phase, protonated YA(n) (n = 1,2) peptides and their dimers were measured with 266 nm photons. The protonated monomers were found to have a fast dissociation channel with an exponential lifetime of ∼200 ns while the protonated dimers show an additional slow dissociation component with a lifetime of ∼2 μs. Laser power dependence measurements enabled us to ascribe the fast channel in the monomer and the slow channel in the dimer to a one-photon process, whereas the fast dimer channel is from a two-photon process. The slow (1 photon) dissociation channel in the dimer was found to result in cleavage of the H-bonds after energy transfer through these H-bonds. In general, the dissociation of these protonated peptides is non-prompt and the decay time was found to increase with the size of the peptides. Quantum RRKM calculations of the microcanonical rate constants also confirmed a statistical nature of the photodissociation processes in the dipeptide monomers and dimers. The classical RRKM expression gives a rate constant as an analytical function of the number of active vibrational modes in the system, estimated separately on the basis of the equipartition theorem. It demonstrates encouraging results in predicting fragmentation lifetimes of protonated peptides. Finally, we present the first experimental evidence for a photo-induced conversion of tyrosine-containing peptides into monocyclic aromatic hydrocarbon along with a formamide molecule both found in space.
- PMID:
- 22239781
- [PubMed - in process]
Single Molecule Dynamics of Lysozyme Processing Distinguishes Linear and Cross-linked Peptidoglycan Substrates.
Abstract
The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field effect transistors through pyrene linker molecules. The substrate driven, hinge bending motions of lysozyme induced dynamic elec-tronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limita-tions of optical quenching or bleaching. For both sub-strates, lysozyme exhibits processive slow turnover rates of 20 - 50 s-1 and rapid 200 - 400 s-1 nonproductive motions. The latter, nonproductive binding events occupy 43% of the enzyme's time in the presence of the cross-linked pepti-doglycan, but only 7% with the linear substrate. Further-more, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate, but appears to sidestep the peptide cross-links to zigzag through the wild-type substrate.
Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors.
Source
Department of Biology, Georgia State University, Atlanta, GA 30303, USA.
Abstract
Clinical inhibitor amprenavir (APV) is less effective on HIV-2 protease (PR(2) ) than on HIV-1 protease (PR(1) ). We solved the crystal structure of PR(2) with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR(1) mutant (PR(1M) ) with substitutions V32I, I47V and V82I that mimic the inhibitor binding site of PR(2) . PR(1M) more closely resembled PR(2) than PR(1) in catalytic efficiency on four substrate peptides and inhibition by APV, while few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR(1M) with APV, DRV, and SQV were compared with available PR(1) and PR(2) complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR(1M) and PR(1) , however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR(1M) . Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, while Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR(1M) and PR(2) relative to the strong hydrogen bonds observed in PR(1) , consistent with 15- and 19-fold worse inhibition, respectively. Overall, PR(1M) partially replicates the specificity of PR(2) and gives insight into drug resistant mutations at residues 32, 47 and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV-2.
Copyright © 2012 The Protein Society.
Investigation of some biologically relevant redox reactions using electrochemical mass spectrometry interfaced by desorption electrospray ionization.
Source
Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Abstract
Recently we have shown that, as a versatile ionization technique, desorption electrospray ionization (DESI) can serve as a useful interface to combine electrochemistry (EC) with mass spectrometry (MS). In this study, the EC/DESI-MS method has been further applied to investigate some aqueous phase redox reactions of biological significance, including the reduction of peptide disulfide bonds and nitroaromatics as well as the oxidation of phenothiazines. It was found that knotted/enclosed disulfide bonds in the peptides apamin and endothelin could be electrochemically cleaved. Subsequent tandem MS analysis of the resulting reduced peptide ions using collision-induced dissociation (CID) and electron-capture dissociation (ECD) gave rise to extensive fragment ions, providing a fast protocol for sequencingpeptides with complicated disulfide bond linkages. Flunitrazepam and clonazepam, a class of nitroaromatic drugs, are known to undergo reduction into amines which was proposed to involve nitroso and N-hydroxyl intermediates. Now in this study, these corresponding intermediate ions were successfully intercepted and their structures were confirmed by CID. This provides mass spectrometric evidence for the mechanism of the nitro to amine conversion process during nitroreduction, an important redox reaction involved in carcinogenesis. In addition, the well-known oxidation reaction of chlorpromazine was also examined. The putative transient one-electron transfer product, the chlorpromazine radical cation (m/z 318), was captured by MS, for the first time, and its structure was also verified by CID. In addition to these observations, some features of the DESI-interfaced electrochemical mass spectrometry were discussed, such as simple instrumentation and the lack of background signal. These results further demonstrate the feasibility of EC/DESI-MS for the study of the biology-relevant redox chemistry and would find applications in proteomics and drug development research.
Structural studies on bovine heart cytochrome c oxidase.
Abstract
Among the X-ray structures of bovine heart cytochrome c oxidase (CcO), reported thus far, the highest resolution is 1.8Å. CcO includes 13 different protein subunits, 7 species of phospholipids, 7 species of triglycerides, 4 redox-active metal sites (Cu(A), heme a (Fe(a)), Cu(B), heme a(3) (Fe(a3))) and 3 redox-inactive metal sites (Mg(2+), Zn(2+) and Na(+)). The effects of various O(2) analogs on the X-ray structure suggest that O(2) molecules are transiently trapped at the Cu(B) site before binding to Fe(a3)(2+) to provide O(2)(-). This provides three possible electron transfer pathways from Cu(B), Fe(a3) and Tyr244 via a water molecule. These pathways facilitate non-sequential 3 electron reduction of the bound O(2)(-) to break the OO bond without releasing active oxygen species. Bovine heart CcO has a proton conducting pathway that includes a hydrogen-bond network and a water-channel which, in tandem, connect the positive side phase with the negative side phase. The hydrogen-bond network forms two additional hydrogen-bonds with the formyl and propionate groups of heme a. Thus, upon oxidation of heme a, the positive charge created on Fe(a) is readily delocalized to the heme peripheral groups to drive proton-transport through the hydrogen-bond network. A peptide bond in the hydrogen-bond network and a redox-coupled conformational change in the water channel are expected to effectively block reverse proton transfer through the H-pathway. These functions of the pathway have been confirmed by site-directed mutagenesis of bovine CcO expressed in HeLa cells. This article is part of a Special Issue entitled: Respiratory Oxidases.
Copyright © 2011. Published by Elsevier B.V.
Broad antiviral activity and crystal structure of HIV-1 fusion inhibitor Sifuvirtide.
Source
Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, China;
Abstract
Sifuvirtide (SFT) is an electrostatically constrained α-helical peptide fusion inhibitor showing potent anti-HIV activity, good safety and pharmacokinetic profiles, and is currently under Phase II clinical trials in China. In this study, we demonstrate its potent and broad anti-HIV activity by using diverse HIV-1 subtypes and variants, including subtypes A, B and C that dominate the AIDS epidemics worldwide, and subtypes B', CRF07_BC and CRF01_AE recombinants that are currently circulating in China, and those possessing cross-resistance to the first and second generation fusion inhibitors. To elucidate its mechanism of action, we determined the crystal structure of SFT in complex with its target NHR peptide (N36), which fully supports our rational inhibitor design and reveals its key motifs and residues responsible for the stability and anti-HIV activity. As anticipated, SFT adopts fully helical conformation stabilized by the multiple engineered salt-bridges. The designing of SFT also provide novel inter-helical salt-bridges and hydrogen bonds that improve the affinity of SFT to NHR trimer. The extra serine residue and acetyl group stabilize α-helicity of the N-terminal portion of SFT while Thr119 serves to stabilize the hydrophobic NHR pocket. In addition, our structure demonstrates that the residues critical for drug resistance, located at positions 37, 38, 41, and 43 of NHR, are irreplaceable for maintaining the stable fusogenic six-helix bundle structure. Our data present important information for developing SFT for clinical use and for designing novel HIV fusion inhibitors.
Cis-trans peptide variations in structurally similar proteins.
Source
INSERM UMR-S 665, Dynamique des Structures et Interactions des Macromolécules Biologiques (DSIMB), Université Denis Diderot-Paris 7, INTS, 6, rue Alexandre Cabanel, 75739, Paris Cedex 15, France.
Abstract
The presence of energetically less favourable cis peptides in protein structures has been observed to be strongly associated with its structural integrity and function. Inter-conversion between the cis and trans conformations also has an important role in the folding process. In this study, we analyse the extent of conservation of cis peptides among similar folds. We look at both the amino acid preferences and local structural changes associated with such variations. Nearly 34% of the Xaa-Proline cis bonds are not conserved in structural relatives; Proline also has a high tendency to get replaced by another amino acid in the trans conformer. At both positions bounding the peptide bond, Glycine has a higher tendency to lose the cis conformation. The cis conformation of more than 30% of β turns of type VIb and IV are not found to be conserved in similar structures. A different view using Protein Block-based description of backbone conformation, suggests that many of the local conformational changes are highly different from the general local structural variations observed among structurally similar proteins. Changes between cis and trans conformations are found to be associated with the evolution of new functions facilitated by local structural changes. This is most frequent in enzymes where new catalytic activity emerges with local changes in the active site. Cis-trans changes are also seen to facilitate inter-domain and inter-protein interactions. As in the case of folding, cis-trans conversions have been used as an important driving factor in evolution.
No comments:
Post a Comment