共查询到20条相似文献，搜索用时 15 毫秒
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Nguyen TN Angkawidjaja C Kanaya E Koga Y Takano K Kanaya S 《Protein science : a publication of the Protein Society》2012,21(4):553-561
Metagenome‐derived LC11‐RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto‐RNase H1). It lacks a C‐terminal tail, which is responsible for hyperstabilization of Sto‐RNase H1. Sto‐RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double‐stranded RNA (dsRNA). To examine whether LC11‐RNase H1 also exhibits both RNase H and dsRNase activities, LC11‐RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11‐RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto‐RNase H1. However, LC11‐RNase H1 did not exhibit dsRNase activity at any condition examined. LC11‐RNase H1 was less stable than Sto‐RNases H1 and its derivative lacking the C‐terminal tail (Sto‐RNase H1ΔC6) by 37 and 13°C in Tm, respectively. To understand the structural bases for these differences, the crystal structure of LC11‐RNase H1 was determined at 1.4 Å resolution. The LC11‐RNase H1 structure is highly similar to the Sto‐RNase H1 structure. However, LC11‐RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA‐phosphate binding pocket, while Sto‐RNase H1 has one groove containing the active site. In addition, LC11‐RNase H1 contains more cavities and buried charged residues than Sto‐RNase H1. We propose that LC11‐RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11‐RNase H1 is less stable than Sto‐RNase H1ΔC6 because of the increase in cavity volume and number of buried charged residues. 相似文献
3.
Hiroyuki Okano Eiko Kanaya Masashi Ozaki Clement Angkawidjaja Shigenori Kanaya 《Protein science : a publication of the Protein Society》2015,24(3):408-419
A metagenome‐derived glycoside hydrolase family 9 enzyme with an N‐terminal immunoglobulin‐like (Ig‐like) domain, leaf‐branch compost (LC)‐CelG, was characterized and its crystal structure was determined. LC‐CelG did not hydrolyze p‐nitrophenyl cellobioside but hydrolyzed CM‐cellulose, indicating that it is endoglucanase. LC‐CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5–9. Its denaturation temperature was 81.4°C, indicating that LC‐CelG is a thermostable enzyme. The structure of LC‐CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29–31%) amino acid sequence identities to LC‐CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC‐CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC‐CelG. Removal of the Ig‐like domain reduced the activity and stability of LC‐CelG by 100‐fold and 6.3°C, respectively. Removal of the Gln40‐ and Asp99‐mediated interactions between the Ig‐like and catalytic domains destabilized LC‐CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig‐like domain contributes to the stabilization of LC‐CelG mainly due to the Gln40‐ and Asp99‐mediated interactions. Because the LC‐CelG derivative lacking the Ig‐like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig‐like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain. 相似文献
4.
Rongjin Guan Sriram Aiyer Marie L. Cote Rong Xiao Mei Jiang Thomas B. Acton Monica J. Roth Gaetano T. Montelione 《Proteins》2017,85(4):647-656
The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc. 相似文献
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Sanjit Kumar Nagendra Singh Mau Sinha Punit Kaur A. Srinivasan Sujata Sharma T. P. Singh 《Acta Crystallographica. Section F, Structural Biology Communications》2009,65(6):635-637
A novel antifungal protein, amaryllin, has been isolated from the underground bulbs of Amaryllis belladonna, purified to homogeneity and crystallized. The protein was extracted using ammonium sulfate fractionation. The purified protein samples indicated a molecular weight of 15 kDa on SDS–PAGE. The protein showed antifungal activity against Aspergillus flavus and Fusarium oxysporum. The N‐terminal sequence of the first 15 amino‐acid residues was determined using Edman degradation and did not show significant sequence identity to any known protein. The protein was crystallized using the hanging‐drop vapour‐diffusion method with 30% PEG 8000 as precipitating agent. The crystals diffracted to 2.7 Å resolution and belonged to the orthorhombic space group I222 or I212121, with unit‐cell parameters a = 48.6, b = 61.9, c = 79.6 Å. The complete sequence and structure determination of amaryllin are in progress. 相似文献
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M. Vinkovic G. Dunn G. E. Wood J. Husain S. P. Wood R. Gill 《Acta Crystallographica. Section F, Structural Biology Communications》2015,71(9):1152-1155
The interaction of momordin, a type 1 ribosome‐inactivating protein from Momordica charantia, with NADP+ and NADPH has been investigated by X‐ray diffraction analysis of complexes generated by co‐crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine–ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide–ribose bond of oxidized NADP+ is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five‐membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors. 相似文献
7.
Katarzyna Kurpiewska Josep Font Marc Ribó Maria Vilanova Krzysztof Lewiński 《Proteins》2009,77(3):658-669
To investigate the structural origin of decreased pressure and temperature stability, the crystal structure of bovine pancreatic ribonuclease A variants V47A, V54A, V57A, I81A, I106A, and V108A was solved at 1.4–2.0 Å resolution and compared with the structure of wild‐type protein. The introduced mutations had only minor influence on the global structure of ribonuclease A. The structural changes had individual character that depends on the localization of mutated residue, however, they seemed to expand from mutation site to the rest of the structure. Several different parameters have been evaluated to find correlation with decrease of free energy of unfolding ΔΔGT, and the most significant correlation was found for main cavity volume change. Analysis of the difference distance matrices revealed that the ribonuclease A molecule is organized into five relatively rigid subdomains with individual response to mutation. This behavior consistent with results of unfolding experiments is an intrinsic feature of ribonuclease A that might be surviving remnants of folding intermediates and reflects the dynamic nature of the molecule. Proteins 2009. © 2009 Wiley‐Liss, Inc. 相似文献
8.
Hyoun Sook Kim Kyung Hee Rhee Byung‐Gyu Kim Dae Gyu Kim Mi Seul Park Hyun‐Jung Kim Sunghoon Kim Byung Woo Han 《Proteins》2013,81(10):1840-1846
Human cytosolic aspartyl‐tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi‐tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Å2 which comprises 16.6% of the monomeric surface area. Our structure reveals the C‐terminal end of the N‐helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N‐helix for the transfer of tRNAAsp to elongation factor 1α. From our analyses of the crystal structure and post‐translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.Proteins 2013; 81:1840–1846. © 2013 Wiley Periodicals, Inc. 相似文献
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Ana Lucia L. R. Zimbardi Matheus P. Pinheiro Marcelo Dias‐Baruffi M. Cristina Nonato 《Acta Crystallographica. Section F, Structural Biology Communications》2010,66(5):542-545
Galectin‐4 is a tandem‐repeat‐type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin‐4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate‐binding specificities of galectin‐4 has been focused on. As an initial step, the N‐terminal carbohydrate‐recognition domain of human galectin‐4 (hGal4‐CRD‐1) has been successfully crystallized using the vapour‐diffusion technique, a complete data set has been collected to 2.2 Å resolution and the structure has been solved by the molecular‐replacement technique. The crystals belonged to space group P6122, with unit‐cell parameters a = b = 71.25, c = 108.66 Å. The asymmetric unit contained one molecule of hGal4‐CRD‐1, with a VM value of 2.34 Å3 Da−1 and a solvent content of 47.51%. 相似文献
10.
The role of the N-terminal polypeptide fragment of the immunoglobulin l-chain in V domain packing stability, and the flexibility of the whole chain was approached by molecular dynamics simulation. The observations were supported by experimental analysis. The N-terminal polypeptide fragment appeared to be the low-stability packing element in the V domain. At moderately elevated temperature it may be replaced at its packing locus by Congo red and then removed by proteolysis. After removal of Congo red by adsorption to (diethylamino)ethyl (DEAE) cellulose, the stability of complete L chain and of L chain devoid of the N-terminal polypeptide fragment were compared. The results indicated that the N-terminal polypeptide fragment plays an essential role in the stability of the V domain. Its removal makes the domain accessible for ANS and Congo red dye binding without heating. The decreased domain stability was registered in particular as increased root mean square (RMS) fluctuation and higher susceptibility to proteolytic attack. The long-range effect was most clearly manifested at 340 K as independent V and C domain fluctuation in the l-chain devoid of the N-terminal polypeptide fragment. This is likely due to the lack of direct connections between the N- and C-termini of the V domain polypeptide. In a complete V domain the connection involves residues 8-12 and 106-110 in particular. Partial or complete disruption of this connection increases the freedom of V domain rotation, while its increased cohesion strengthens the coupling of the V and C domains, making the whole L chain less flexible. 相似文献
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The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage. 相似文献
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Junko Onishi Molay Kumar Roy Lekh Raj Juneja Yasuo Watanabe Youichi Tamai 《Journal of peptide science》2008,14(9):1032-1038
Model studies have shown that peptides derived from the N-terminal region of bovine lactoferrin (Lf-B) exhibit antitumor activity against certain cell lines. This activity is due primarily to the peptides' apoptogenic effect. Several reports indicate that cationic residues clustered in two regions of the peptide sequence can be shuffled into one region and thereby increase cytotoxic activity, although the mechanism of this enhanced cytotoxic effect has not been clarified. In this paper, we considered several parameters that determine the mode of cell death after exposure to a native Lf-B derived peptide (Pep1, residues 17-34), and a modified peptide (mPep1) wherein the cationic residues of Pep1 are clustered in a single region of its helical structure. We found that the cytotoxic activity of mPep1 was about 9.6 fold-higher than that of Pep1 against HL-60 cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay. In investigating the expression of phosphatidylserine, we observed that the native peptide (Pep1) caused both apoptotic cell death and necrotic cell death, depending on the concentration of the peptide. In contrast, the action of mPep1 was exclusively characteristic of necrotic cell death. This observation was further confirmed by agarose gel electrophoresis, in which clear ladder-like DNA bands were observed from cells exposed to Pep1, whereas DNA from cells treated with mPep1 produced a smeared pattern. We extended the study by investigating the release of mitochondrial cytochrome c into the cytosol, and the activation of caspase-3; both peptides caused the release of cytochrome c into the cytosol, and the activation of caspase-3.These results suggest that Pep1 may kill cancer cells by activating an apoptosis-inducing pathway, whereas mPep1 causes necrotic cell death by destroying cellular membrane structure notwithstanding sharing some cellular events with apoptotic cell death. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
13.
Terzyan S Wang CS Downs D Hunter B Zhang XC 《Protein science : a publication of the Protein Society》2000,9(9):1783-1790
Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1-538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 A resolution. The crystal form belongs to space group P2(1)2(1)2(1) with one monomer per asymmetric unit, and the protein shows an alpha/beta hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115-125 and 270-285). 相似文献
14.
Peptide C‐terminal N‐alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C‐terminal N‐alkyl amides using the well‐known Fukuyama N‐alkylation reaction on a standard resin commonly used for the synthesis of peptide C‐terminal primary amides, the peptide amide linker‐polyethylene glycol‐polystyrene (PAL‐PEG‐PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N‐alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N‐alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N‐alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N‐alkyl amides. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 715–722, 2011. 相似文献
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The AP7 and AP24 proteins represent a class of mineral-interaction polypeptides that are found in the aragonite-containing nacre layer of mollusk shell (H. rufescens). These proteins have been shown to preferentially interfere with calcium carbonate mineral growth in vitro. It is believed that both proteins play an important role in aragonite polymorph selection in the mollusk shell. Previously, we demonstrated the 1-30 amino acid (AA) N-terminal sequences of AP7 and AP24 represent mineral interaction/modification domains in both proteins, as evidenced by their ability to frustrate calcium carbonate crystal growth at step edge regions. In this present report, using free N-terminal, C(alpha)-amide capped synthetic polypeptides representing the 1-30 AA regions of AP7 (AP7-1 polypeptide) and AP24 (AP24-1 polypeptide) and NMR spectroscopy, we confirm that both N-terminal sequences possess putative Ca (II) interaction polyanionic sequence regions (2 x -DD- in AP7-1, -DDDED- in AP24-1) that are random coil-like in structure. However, with regard to the remaining sequences regions, each polypeptide features unique structural differences. AP7-1 possesses an extended beta-strand or polyproline type II-like structure within the A11-M10, S12-V13, and S28-I27 sequence regions, with the remaining sequence regions adopting a random-coil-like structure, a trait common to other polyelectrolyte mineral-associated polypeptide sequences. Conversely, AP24-1 possesses random coil-like structure within A1-S9 and Q14-N16 sequence regions, and evidence for turn-like, bend, or loop conformation within the G10-N13, Q17-N24, and M29-F30 sequence regions, similar to the structures identified within the putative elastomeric proteins Lustrin A and sea urchin spicule matrix proteins. The similarities and differences in AP7 and AP24 N-terminal domain structure are discussed with regard to joint AP7-AP24 protein modification of calcium carbonate growth. 相似文献
17.
Kinga Gerber Andr Schiefner Peter Seige Kay Diederichs Winfried Boos Wolfram Welte 《Acta Crystallographica. Section D, Structural Biology》2004,60(3):531-533
Aes belongs to the family of hormone‐sensitive lipases and has acetyl‐esterase activity. It is also known to control maltose uptake through interaction with MalT, the central regulator of the Escherichia coli maltose system. Aes was crystallized as an N‐terminally His6‐tagged protein both in the native form and with selenomethionine substitution. Crystals grew in both cases in space group R32 to dimensions of about 0.2 × 0.15 × 0.05 mm (native His6‐Aes) and about 0.5 × 0.3 × 0.1 mm (SeMet‐His6‐Aes). A native data set has been obtained at 2.4 Å resolution; the selenomethionine‐substituted Aes crystals diffracted to 3.0 Å resolution. 相似文献
18.
Caroline Proulx Falko Noë Stan Yoo Michael D. Connolly Ronald N. Zuckermann 《Peptide Science》2016,106(5):726-736
A novel approach to sequentially degrade peptoid N‐terminal N‐(substituted)glycine residues on the solid‐phase using very mild conditions is reported. This method relies on the treatment of resin‐bound, bromoacetylated peptoids with silver perchlorate in THF, leading to an intramolecular cyclization reaction to liberate the terminal residue as a N‐substituted morpholine‐2,5‐dione, resulting in a truncated peptoid upon hydrolysis and a silver bromide byproduct. Side‐chain functional group tolerance is explored and reaction kinetics are determined. In a series of pentapeptoids possessing variable, non‐nucleophilic side‐chains at the second position (R2), we demonstrate that sequential N‐terminal degradation of the first two residues proceeds in 87% and 74% conversions on average, respectively. We further demonstrate that the degradation reaction is selective for peptoids, and represents substantial progress toward a mild, iterative sequencing method for peptoid oligomers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 726–736, 2016. 相似文献
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Mohamed A. Eldeeb Richard P. Fahlman Mohamed A. Ragheb Mansoore Esmaili 《BioEssays : news and reviews in molecular, cellular and developmental biology》2019,41(11)
The N‐end rule denotes the relationship between the identity of the amino‐terminal residue of a protein and its in vivo half‐life. Since its discovery in 1986, the N‐end rule has generally been described by a defined set of rules for determining whether an amino‐terminal residue is stabilizing or not. However, recent studies are revealing that this N‐end rule (or N‐degron concept) is less straightforward than previously appreciated. For instance, it is unveiled that N‐terminal acetylation of N‐terminal residues may create a degradation signal (Ac‐degron) that promotes the degradation of target proteins. A recent high‐throughput dissection of degrons in yeast proteins amino termini intriguingly suggested that the hydrophobicity of amino‐terminal residues—but not the N‐terminal acetylation status—may be the indispensable feature of amino‐terminal degrons. Herein, these recent advances in N‐terminal acetylation and the complexity of N‐terminal degradation signals in the context of the N‐degron pathway are analyzed. 相似文献
20.
We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications. 相似文献