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Histidine Residues (histidine + residue)
Selected AbstractsInvolvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilizationFEBS JOURNAL, Issue 13 2000Vincent Monchois Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the ,-amylase family (glycoside hydrolase family 13), which all have a (,/,)8 barrel structure. In the glucansucrases, however, the ,-helix and ,-strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In ,-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis,Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (,/,)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which ,1,3-linked glucosyl units are joined sequentially to maltose. [source] Structure,Activity Relationship Studies in Single-Site Esterase Peptide DendrimersISRAEL JOURNAL OF CHEMISTRY, Issue 1 2009Sacha Javor We recently reported on peptide dendrimers with a single catalytic site at the dendrimer core catalyzing the hydrolysis of acetoxy- and butyryloxy-pyrene trisulfonate 1a/b in aqueous buffer with Michaelis,Menten kinetics. Substrate binding is mediated by a pair of protonated arginine or histidine residues in the first generation branch, and esterolysis is performed by the imidazole side-chain of a histidine residue in the core acting as a general base or nucleophile. Herein we report on a structure,activity relationship study searching for an optimal combination between amino acid sequence and catalytic machinery. Installation of histidine residues onto the aromatic dendrimer framework "R" leads to 10-fold higher rate acceleration up to kcat/kuncat = 1.5 * 103 at pH 5.5 with dendrimers RG3H (AcYT)8 (BWG)4 (BHS)2BHS and RMG3H (AcYT)8(BWG)4(BHSG)2BHS (one-letter codes for L -amino acids; Ac = acetyl, B = L -2,3-diaminopropionic acid branching point, C-terminus is amide -CONH2). These dendrimers reach the compactness of a native folded protein. [source] Histidine and not tyrosine is required for the Peroxide-induced formation of haem to protein cross-linked myoglobinIUBMB LIFE, Issue 8-9 2007Brandon J. Reeder Abstract Peroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An understanding of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications associated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 - 10541). Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr103 , Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein's capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome. [source] Evaluation of the metal binding properties of a histidine-rich fusogenic peptide by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2003Andrea Sinz Abstract Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used to investigate metal ion interactions of the 18 amino acid peptide fragment B18 (LGLLLRHLRHHSNLLANI), derived from the membrane-associated protein bindin. The peptide sequence B18 represents the minimal membrane-binding motif of bindin and resembles a putative fusion peptide. The histidine-rich peptide has been shown to self-associate into distinct supramolecular structures, depending on the presence of Zn2+ and Cu2+. We examined the binding of B18 to the metal ions Cu2+, Zn2+, Mg2+, Ca2+, Mn2+ and La3+. For Cu2+, we compared the metal binding affinities of the wild-type B18 peptide with those of its mutants in which one, two or three histidine residues have been replaced by serines. Upon titration of B18 with Cu2+ ions, we found sequential binding of two Cu2+ ions with dissociation constants of ,34 and ,725 µM. Mutants of B18, in which one histidine residue is replaced by serine, still exhibit sequential binding of two copper ions with affinities for the first Cu2+ ion comparable to that of wild-type B18 peptide, but with a greatly reduced affinity for the second Cu2+ ion in mutants H112S and H113S. For mutants in which two histidines are replaced by serines, the affinity for the first Cu2+ ion is reduced ,3,10 times in comparison with B18. The mutant in which all three histidine residues are replaced by serines exhibits an ,14-fold lower binding for the first Cu2+ ion compared with B18. For the other metal ions under investigation (Zn2+, Mg2+, Ca2+, Mn2+ and La3+), a modest affinity to B18 was detected binding to the peptide in a 1 : 1 stoichiometry. Our results show a high affinity of the wild-type fusogenic peptide B18 for Cu2+ ions whereas the Zn2+ affinity was found to be comparable to that of other di- and trivalent metal ions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Structure and heme binding properties of Escherichia coli O157:H7 ChuXPROTEIN SCIENCE, Issue 4 2009Michael D. L. Suits Abstract For many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram-negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and utilization of heme as an iron source. While the complement of proteins responsible for heme internalization has been determined in these organisms, the fate of heme once it has reached the cytoplasm has only recently begun to be resolved. Here we report the first crystal structure of ChuX, a member of the conserved heme utilization operon from pathogenic E. coli O157:H7 determined at 2.05 Ĺ resolution. ChuX forms a dimer which remarkably given low sequence homology, displays a very similar fold to the monomer structure of ChuS and HemS, two other heme utilization proteins. Absorption spectral analysis of heme reconstituted ChuX demonstrates that ChuX binds heme in a 1:1 manner implying that each ChuX homodimer has the potential to coordinate two heme molecules in contrast to ChuS and HemS where only one heme molecule is bound. Resonance Raman spectroscopy indicates that the heme of ferric ChuX is composed of a mixture of coordination states: 5-coordinate and high-spin, 6-coordinate and low-spin, and 6-coordinate and high-spin. In contrast, the reduced ferrous form displays mainly a 5-coordinate and high-spin state with a minor contribution from a 6-coordinate and low-spin state. The ,Fe-CO and ,C-O frequencies of ChuX-bound CO fall on the correlation line expected for histidine-coordinated hemoproteins indicating that the fifth axial ligand of the ferrous heme is the imidazole ring of a histidine residue. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuX to probe the heme binding sites and dimer interface. Spectral analysis of ChuX and mutants suggests involvement of H65 and H98 in heme coordination as mutations of both residues were required to abolish the formation of the hexacoordination state of heme-bound ChuX. [source] The subcellular distribution of the Arabidopsis histidine phosphotransfer proteins is independent of cytokinin signalingTHE PLANT JOURNAL, Issue 3 2010Jayson A. Punwani Summary Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway. [source] Ion channel activity of transmembrane segment 6 of Escherichia coli proton-dependent manganese transporterBIOPOLYMERS, Issue 8 2010uková Abstract Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure,function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His211 were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure,function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718,726, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Model peptide-based system used for the investigation of metal ions binding to histidine-containing polypeptidesBIOPOLYMERS, Issue 6 2010Manuela Murariu Abstract The reaction of histidine-containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI,MS) and circular dichroism (CD). The analysis of CD-spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal-bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497,508, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2 -producing ligninolytic enzymeFEBS JOURNAL, Issue 21 2006Patricia Ferreira Aryl-alcohol oxidase provides H2O2 for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans, and characterized in terms of their steady-state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl-alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin-adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finaly, L315 affects the enzyme kcat, although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl-alcohol oxidase active site residues. [source] Solution structure of a zinc-finger domain that binds to poly-ADP-riboseGENES TO CELLS, Issue 2 2010Shin Isogai Poly-ADP-ribosylation is a unique post-translational modification that controls various nuclear events such as repair of DNA single-strand breaks. Recently, the protein containing the poly-ADP-ribose (pADPr)-binding zinc-finger (PBZ) domain was shown to be a novel AP endonuclease and involved in a cell cycle checkpoint. Here, we determined the three-dimensional structure of the PBZ domain from Drosophila melanogaster CG1218-PA using NMR spectroscopy. The domain folds into a C2H2-type zinc-finger structure in an S configuration, containing a characteristic loop between the zinc-coordinating cysteine and histidine residues. This is distinct from the structure of other C2H2-type zinc fingers. NMR signal changes that occur when pADPr binds to the PBZ domains from CG1218-PA and human checkpoint with FHA (forkhead-associated) and ring finger (CHFR) and mutagenesis suggest that a surface relatively well conserved among PBZ domains may serve as a major interface with pADPr. [source] Detection of tmRNA-mediated trans-translation products in Bacillus subtilisGENES TO CELLS, Issue 3 2002Ai Fujihara Background: Bacterial tmRNA (10Sa RNA) is involved in a trans -translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes. To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo trans -translation. Results: The wild-type tmRNA gene (ssrA) in the genome was replaced by a variant ssrA encoding a tag-peptide sequence containing six histidine residues (His-tag) and two aspartic acids at the C-terminus. The His-tagged proteins that accumulated in the cells without degradation were fractionated by Ni2+ -NTA column and gel electrophoresis and were detected by Western blotting with an anti-His-tag antibody. The results showed that the trans -translation occurred more frequently at a high temperature (50 °C) than at a low temperature (37 °C). Two-dimensional (2D) gel electrophoresis of the products revealed many distinct spots, which represent specific target proteins for the trans -translation reaction. Furthermore, the 2D gel patterns of the products from cells cultured at high and low temperatures were apparently different. Several tagged proteins were identified by the N-terminal amino acid sequences of the products. Conclusion:Trans -translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures. [source] Structure,Activity Relationship Studies in Single-Site Esterase Peptide DendrimersISRAEL JOURNAL OF CHEMISTRY, Issue 1 2009Sacha Javor We recently reported on peptide dendrimers with a single catalytic site at the dendrimer core catalyzing the hydrolysis of acetoxy- and butyryloxy-pyrene trisulfonate 1a/b in aqueous buffer with Michaelis,Menten kinetics. Substrate binding is mediated by a pair of protonated arginine or histidine residues in the first generation branch, and esterolysis is performed by the imidazole side-chain of a histidine residue in the core acting as a general base or nucleophile. Herein we report on a structure,activity relationship study searching for an optimal combination between amino acid sequence and catalytic machinery. Installation of histidine residues onto the aromatic dendrimer framework "R" leads to 10-fold higher rate acceleration up to kcat/kuncat = 1.5 * 103 at pH 5.5 with dendrimers RG3H (AcYT)8 (BWG)4 (BHS)2BHS and RMG3H (AcYT)8(BWG)4(BHSG)2BHS (one-letter codes for L -amino acids; Ac = acetyl, B = L -2,3-diaminopropionic acid branching point, C-terminus is amide -CONH2). These dendrimers reach the compactness of a native folded protein. [source] Evaluation of the metal binding properties of a histidine-rich fusogenic peptide by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2003Andrea Sinz Abstract Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used to investigate metal ion interactions of the 18 amino acid peptide fragment B18 (LGLLLRHLRHHSNLLANI), derived from the membrane-associated protein bindin. The peptide sequence B18 represents the minimal membrane-binding motif of bindin and resembles a putative fusion peptide. The histidine-rich peptide has been shown to self-associate into distinct supramolecular structures, depending on the presence of Zn2+ and Cu2+. We examined the binding of B18 to the metal ions Cu2+, Zn2+, Mg2+, Ca2+, Mn2+ and La3+. For Cu2+, we compared the metal binding affinities of the wild-type B18 peptide with those of its mutants in which one, two or three histidine residues have been replaced by serines. Upon titration of B18 with Cu2+ ions, we found sequential binding of two Cu2+ ions with dissociation constants of ,34 and ,725 µM. Mutants of B18, in which one histidine residue is replaced by serine, still exhibit sequential binding of two copper ions with affinities for the first Cu2+ ion comparable to that of wild-type B18 peptide, but with a greatly reduced affinity for the second Cu2+ ion in mutants H112S and H113S. For mutants in which two histidines are replaced by serines, the affinity for the first Cu2+ ion is reduced ,3,10 times in comparison with B18. The mutant in which all three histidine residues are replaced by serines exhibits an ,14-fold lower binding for the first Cu2+ ion compared with B18. For the other metal ions under investigation (Zn2+, Mg2+, Ca2+, Mn2+ and La3+), a modest affinity to B18 was detected binding to the peptide in a 1 : 1 stoichiometry. Our results show a high affinity of the wild-type fusogenic peptide B18 for Cu2+ ions whereas the Zn2+ affinity was found to be comparable to that of other di- and trivalent metal ions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Mark R. Contarino Abstract As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1,mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15,nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain. Copyright © 2006 John Wiley & Sons, Ltd. [source] New insights into intracellular lipid binding proteins: The role of buried waterPROTEIN SCIENCE, Issue 10 2002Christian Lücke Abstract The crystal structures of most intracellular lipid binding proteins (LBPs) show between 5 and 20 internally bound water molecules, depending on the presence or the absence of ligand inside the protein cavity. The structural and functional significance of these waters has been discussed for several LBPs based on studies that used various biophysical techniques. The present work focuses on two very different LBPs, heart-type fatty acid binding protein (H-FABP) and ileal lipid binding protein (ILBP). Using high-resolution nuclear magnetic resonance spectroscopy, certain resonances belonging to side-chain protons that are located inside the water-filled lipid binding cavity were observed. In the case of H-FABP, the pH- and temperature-dependent behavior of selected side-chain resonances (Ser82 OgH and the imidazole ring protons of His93) indicated an unusually slow exchange with the solvent, implying that the intricate hydrogen-bonding network of amino-acid side-chains and water molecules in the protein interior is very rigid. In addition, holo H-FABP appeared to display a reversible self-aggregation at physiological pH. For ILBP, on the other hand, a more solvent-accessible protein cavity was deduced based on the pH titration behavior of its histidine residues. Comparison with data from other LBPs implies that the evolutionary specialization of LBPs for certain ligand types was not only because of mutations of residues directly involved in ligand binding but also to a refinement of the internal water scaffold. [source] Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2006Aniel Sánchez Abstract A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3 - and d0 -acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N -acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R,+,H,,,1) that are neither retained nor analyzed, whereas the multiply charged peptides (R,+,H>1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae. [source] Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2005Fagen Zhang The reactions of two model mutagenic and carcinogenic alkylating agents, N -methyl- N -nitrosourea (MNU) and methyl methanesulfonate (MMS), with proteins and deoxynucleosides in vitro, were investigated. The protein work used an approach involving trypsin digestion and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This technique permitted identification of the specific location of protein adduction by both MNU and MMS with commercial apomyoglobin and human hemoglobin, under physiological conditions. MNU treatment resulted in predominantly carbamoylation adducts on the proteins, but in contrast only methylated protein adducts were found following treatment with MMS. Further analyses, using TurboSequest®, and the Scoring Algorithm for Spectral Analysis (SALSA), revealed that MNU carbamoylation was specific for modification of either the N-terminal valine or the free amino group in lysine residues of apomyglobin and human hemoglobin. However, MMS methylation modified the N-terminal valine and histidine residues of the proteins. Despite their clear differences in protein modifications, MNU and MMS formed qualitatively the same methylated deoxynucleoside adduct profiles with all four deoxynucleosides in vitro under physiological conditions. In light of their different biological potencies, where MMS is considered a ,super clastogen' while MNU is a ,super mutagen', these differences in reaction products with proteins vs. deoxynucleosides may indicate that these two model alkylating agents work via different mechanisms to produce their mutagenic and carcinogenic effects. Copyright © 2005 John Wiley & Sons, Ltd. [source] Model peptide-based system used for the investigation of metal ions binding to histidine-containing polypeptidesBIOPOLYMERS, Issue 6 2010Manuela Murariu Abstract The reaction of histidine-containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI,MS) and circular dichroism (CD). The analysis of CD-spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal-bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497,508, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Crystal structure of bovine 3-hydroxyanthranilate 3,4-dioxygenase,,BIOPOLYMERS, Issue 12 2009Ivica, ilovi Abstract 3-Hydroxyanthranilate 3,4-dioxygenase, the enzyme that catalyzes the conversion of 3-hydroxyanthranilate to quinolinic acid, has been extracted and purified from bovine kidney, crystallized and its structure determined at 2.5 Ĺ resolution. The enzyme, which crystallizes in the triclinic P1 space group, is a monomer, characterized by the so-called cupin fold. The monomer of the bovine enzyme mimics the dimer present in lower species, such as bacteria and yeast, since it is composed of two domains: one of them is equivalent to one monomer, whilst the second domain corresponds to only a portion of it. The active site consists of an iron ion coordinated by two histidine residues, one glutamate and an external ligand, which has been interpreted as a solvent molecule. It is contained in the N-terminal domain, whilst the function of the C-terminal domain is possibly structural. The catalytic mechanism very likely has been conserved through all species, since the positions of all residues considered relevant for the reaction are present from bacteria to humans. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1189,1195, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] ,-sheet folding of 11-kDa fibrillogenic polypeptide is completely aggregation drivenBIOPOLYMERS, Issue 4 2007Natalya I. Topilina Abstract A de novo polypeptide GH6[(GA)3GY(GA)3GE]8GAH6 (YE8) was designed and genetically engineered to form antiparallel ,-strands of GAGAGA repeats. Modulation of pH enables control of solubility, folding, and aggregation of YE8 by control of the overall polypeptide charge, a consequence of the protonation or deprotonation of the glutamic acid and histidine residues. YE8 exhibits all the major properties of a fibrillogenic protein providing an excellent model for detailed study of the fibrillation. At neutral pH, YE8 is soluble in disordered form, yet at pH 3.5 folds into a predominantly ,-sheet conformation that is fibrillogenic. Atomic force microscopy and transmission electron microscopy indicated the formation of fibrillar aggregates on well-defined, hydrophobic surfaces. The ,-sheet folding of YE8 exhibited a lag phase that could be eliminated by seeding or stirring. The strong dependence of lag time on polypeptide concentration established the limiting step in aggregation as initiation of ,-sheet folding. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 261,264, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Characterization of the polyanion-induced molten globule-like state of cytochrome cBIOPOLYMERS, Issue 2 2007Erik Sedlák Abstract Cytochrome c (cyt c) undergoes a poly(vinylsulphate) (PVS)-induced transition at slightly acidic pH into a molten globule-like state that resembles the effect that negatively charged membrane surfaces have on this protein. In this work, the thermodynamic properties of the molten globule-like state of cyt c in complex with PVS are studied using differential scanning calorimetry, circular dichroism, fluorescence, and absorbance spectroscopy. The temperature-induced transition of the molten globule-like state of cyt c in the complex with PVS is characterized by a significantly lower calorimetric enthalpy than in the "typical" molten globule state of cyt c, i.e. free protein at pH 2.0 in high ionic strength. Moreover, the thermally-denatured state of cyt c in the complex at pH < 6 contains nearly 50% of the native secondary structure. The dependence of the transition temperature on the pH indicates a role for histidine residues in the destabilization of the cyt c structure in the PVS complex and in stabilization of the denatured state with the residual secondary structure. A comparison of the effects of small anions and polyanions demonstrates the importance of cooperativity among the anions in the destabilization of cyt c. Predictably, other hydrophilic flexible polyanions such as heparin, polyglutamate, and polyadenylate also have a destabilizing effect on the structure of cyt c. However, a correlation between the properties of the polyanions and their effect on the protein stability is still unclear. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 119,126, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Separation of Pure and Immunoreactive Virus-Like Particles Using Gel Filtration Chromatography Following Immobilized Metal Ion Affinity ChromatographyBIOTECHNOLOGY PROGRESS, Issue 2 2001Yu-Shen Cheng A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni2+) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm3. However, the former method can process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely obtained from a 500 mL Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their respective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus. [source] The ybeY protein from Escherichia coli is a metalloproteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2005Chenyang Zhan The three-dimensional crystallographic structure of the ybeY protein from Escherichia coli (SwissProt entry P77385) is reported at 2.7,Ĺ resolution. YbeY is a hypothetical protein that belongs to the UPF0054 family. The structure reveals that the protein binds a metal ion in a tetrahedral geometry. Three coordination sites are provided by histidine residues, while the fourth might be a water molecule that is not seen in the diffraction map because of its relatively low resolution. X-ray fluorescence analysis of the purified protein suggests that the metal is a nickel ion. The structure of ybeY and its sequence similarity to a number of predicted metal-dependent hydrolases provides a functional assignment for this protein family. The figures and tables of this paper were prepared using semi-automated tools, termed the Autopublish server, developed by the New York Structural GenomiX Research Consortium, with the goal of facilitating the rapid publication of crystallographic structures that emanate from worldwide Structural Genomics efforts, including the NIH-funded Protein Structure Initiative. [source] Novel Probes Showing Specific Fluorescence Enhancement on Binding to a Hexahistidine TagCHEMISTRY - A EUROPEAN JOURNAL, Issue 26 2008Mie Kamoto Abstract The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel,nitrilotriacetic acid (Ni2+,NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal,NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal,NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni2+ and Co2+ were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni2+ and Co2+ complexes of NTACs in pH,7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)6 -Tyr-NH2; Tyr was included to allow convenient concentration measurement) in pH,7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co2+ complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin,I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide. [source] Synthesis and Esterolytic Activity of Catalytic Peptide DendrimersCHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2004David Lagnoux Abstract Peptide dendrimers were prepared by solid-phase peptide synthesis. Monomeric dendrimers were first obtained by assembly of a hexapeptide sequence containing alternate standard , -amino acids with diamino acids as branching units. The monomeric dendrimers were then dimerized by disulfide-bridge formation at the core cysteine. The synthetic strategy is compatible with functional amino acids and different diamino acid branching units. Peptide dendrimers composed of the catalytic triad amino acids histidine, aspartate, and serine catalyzed the hydrolysis of N -methylquinolinium salts when the histidine residues were placed at the outermost position. The dendrimer-catalyzed hydrolysis of 7-isobutyryl- N -methylquinolinium followed saturation kinetics with a rate constant of catalysis/rate constant without catalysis (kcat/kuncat) value of 3350 and a rate constant of catalysis/Michaelis constant (kcat/KM) value 350-fold larger than the second-order rate constant of the 4-methylimidazole-catalyzed reaction; this corresponds to a 40-fold rate enhancement per histidine side chain. Catalysis can be attributed to the presence of histidine residues at the surface of the dendrimers. [source] | |||||||||||||||||||||||||||||||