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Motion of spin-labeled side chains in T4 lysozyme:: Effect of side chain structure

BIOCHEMISTRY 1999年第10期38卷 2947-2955页

作者： Mchaourab, HS Kálai, T Hideg, K Hubbell, WL Univ Calif Los Angeles Jules Stein Eye Inst Los Angeles CA 90095 USA Univ Calif Los Angeles Dept Chem & Biochem Los Angeles CA 90095 USA Univ Pecs Inst Organ & Med Chem H-7643 Pecs Hungary

Previous studies have shown that the mobility of nitroxide side chains in a protein, inferred from the electron paramagnetic resonance (EPR) spectra, can be used to classify particular sites as helix surface sites, tertiary contact sites, buried sites, or loop sites. In addition, the sequence dependence of mobility can identify regular secondary structure. However, in the most widely used side chain, an apparent interaction of the nitroxide ring with the protein at some helix surface sites gives rise to EPR spectra degenerate with those at tertiary contact sites. In the present study, we use selected sites in T4 lysozyme to evaluate novel nitroxide side chains designed to resolve this degeneracy. The results indicate that the reagent 3-(methanesulfonylthiomethyl)-2,2,5,5-tetramethylpyrrolidin-1-yloxy reacts with cysteine to give a nitroxide side chain that has a high contrast in mobility between helix surface and tertiary contact sites, effectively resolving the degeneracy. The reagent 3-(iodomercuriomethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy reacts with cysteine to provide a mercury-linked nitroxide that also shows reduced interaction with the protein at most helix surface sites. Thus, these new side chains may be the preferred choices for structure determination using site-directed spin labeling.

关键词：氨基酸取代/遗传学精氨酸/遗传学天冬氨酸/遗传学细菌噬菌体T4/酶学结合部位/遗传学电子自旋共振谱学模型分子胞壁质酶/化学胞壁质酶/遗传学诱变定点氧化氮类/化学肽碎片/化学肽碎片/遗传学蛋白质结构二级蛋白质结构三级自旋标记物构效关系缬氨酸/遗传学

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Microsecond folding of the cold shock protein measured by a pressure-jump technique

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BIOCHEMISTRY 1999年第10期38卷 2882-2891页

作者： Jacob, M Holtermann, G Perl, D Reinstein, J Schindler, T Geeves, MA Schmid, FX Univ Bayreuth Biochem Lab D-95440 Bayreuth Germany Max Planck Inst Mol Physiol D-44139 Dortmund Germany

A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding. In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 mu s and then held constant. Thus, unfolding and refolding reactions in the time range from 70 mu s to 70 s can be followed with this technique. Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions. We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region. For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains. The Phe27Ala mutation affected the rate of unfolding only;the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding. Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.

关键词：丙氨酸/化学丙氨酸/遗传学氨基酸取代/遗传学芽孢杆菌枯草细菌蛋白质类/化学细菌蛋白质类/遗传学载体蛋白质类/化学载体蛋白质类/遗传学胍热休克蛋白质类动力学苯丙氨酸/化学苯丙氨酸/遗传学压力蛋白质折叠热力学时间因素黏度

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Species-specific inhibition of inosine 5′-monophosphate dehydrogenase by mycophenolic acid

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BIOCHEMISTRY 1999年第46期38卷 15388-15397页

作者： Digits, JA Hedstrom, L Brandeis Univ Dept Biochem Waltham MA 02454 USA

IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor of mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits IMPDH by binding in the nicotinamide half of the dinucleotide site and trapping the covalent intermediate E-XMP*. The MPA binding site of resistant IMPDH from the parasite Tritrichomonas foetus contains two residues that differ from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Arg and Gin, respectively, in the human type 2 enzyme. We characterized three mutants of T, foetus IMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species selectivity of MPA. The mutation of Lys310Arg causes a 10-fold decrease in the K-i for MPA inhibition and a 8-13-fold increase in the K-m values for IMP and NAD(+). The mutation of Glu431Gln causes a 6-fold decrease in the K-i for MPA. The double mutant displays a 20-fold increase in sensitivity to MPA. Pre-steady-state kinetics were performed to obtain rates of hydride transfer, NADH release, and hydrolysis of E-XMP* for the mutant IMPDHs. The Lys310Arg mutation results in a 3-fold increase in the accumulation level of E-XMP*, while the Glu431Gln mutation has only a minimal effect on the kinetic mechanism. These experiments show that 20 of the 450-fold difference in sensitivity between the T. foetus and human IMPDHs derive from the residues in the MPA binding site. Of this, 3-fold can be attributed to a change in kinetic mechanism. In addition, we measured MPA binding to enzyme adducts with 6-Cl-IMP and EICARMP. Neither of these adducts proved to be a good model for E-XMP*.

关键词：氨基酸取代/遗传学结合竞争性/遗传学酶抑制剂/药理学 IMP脱氢酶/拮抗剂和抑制剂 IMP脱氢酶/化学 IMP脱氢酶/遗传学 IMP脱氢酶/代谢肌苷一磷酸/代谢动力学诱变定点霉酚酸/药理学原虫蛋白质类/拮抗剂和抑制剂原虫蛋白质类/化学原虫蛋白质类/遗传学原虫蛋白质类/代谢重组蛋白质类/化学合成重组蛋白质类/化学重组蛋白质类/分离和提纯核糖核苷酸类/代谢种特异性光谱法荧光分光光度法三毛滴虫胚胎/酶学动物人类

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Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: Evaluation of reversible unfolding conditions

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BIOCHEMISTRY 1999年第46期38卷 15380-15387页

作者： Chen, GQ Gouaux, E Columbia Univ Dept Biochem & Mol Biophys New York NY 10032 USA

Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1,2-dimristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles, To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)], sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.

关键词：氨基酸取代/遗传学噬菌调理素/化学合成噬菌调理素/化学噬菌调理素/遗传学噬菌调理素/代谢胆酸类/代谢洗涤剂/代谢二肉豆蔻磷脂酰胆碱/代谢胍盐杆菌盐制品氢离子浓度微胶粒点突变蛋白质变性/遗传学蛋白质折叠重组融合蛋白质类/化学合成重组融合蛋白质类/代谢十二烷硫酸钠/化学溶液分光光度法紫外线温度尿素

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Characterization of determinants for the specificity of Arabidopsis thioredoxins h in yeast complementation

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第41期275卷 31641-31647页

作者： Bréhélin, C Mouaheb, N Verdoucq, L Lancelin, JM Meyer, Y Univ Perpignan UMR CNRS 5096 Lab Genome & Dev Plantes F-66860 Perpignan France Univ Lyon 1 Lab Resonance Magnet Nucl Biomol CNRS F-69622 Villeurbanne France Ecole Super Chim Phys & Elect F-69622 Villeurbanne France

The disruption of the two thioredoxin genes in Saccharomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H2O2 tolerance, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce methionine sulfoxide assimilation and restore a normal cell cycle. In addition AtTRX2 also confers growth on sulfate but no H2O2 tolerance, In contrast, AtTRX3 does not confer growth on sulfate but induces H2O2 tolerance. We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H2O2 tolerance is located in the N-terminal part of AtTRX3. In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipotent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins. After duplications each member evolved by losing or masking some of the determinants.

关键词：氨基酸序列氨基酸取代/遗传学拟南芥属/酶学拟南芥属/遗传学结合部位印迹法蛋白质细胞周期进化分子遗传互补测验过氧化氢/药理学甲硫氨酸/类似物和衍生物甲硫氨酸/代谢模型分子分子序列数据突变氧化还原表型蛋白质构象重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/代谢酵母菌酿酒/细胞学酵母菌酿酒/药物作用酵母菌酿酒/酶学酵母菌酿酒/遗传学序列比对底物特异性硫酸盐类/代谢

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Association between expression of reproductive seasonality and alleles of the gene for Mel_1a receptor in the ewe

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BIOLOGY OF REPRODUCTION 2000年第4期62卷 1096-1101页

作者： Pelletier, J Bodin, L Hanocq, E Malpaux, B Teyssier, J Thimonier, J Chemineau, P INRA PRMD Equipe Neuroendocrinol Sexuelle CNRSURA 1291 F-37380 Nouzilly France Stn Ameliorat Genet Anim F-31326 Castanet Tolosan France INRA ENSA Unite Zootechnie Mediterraneenne F-34060 Montpellier France

To determine whether a link exists between reproductive seasonality and the structure of the gene for melatonin receptor Mel(1a), the latter was studied in two groups of Merinos d'Arles (MA) ewes previously chosen for their genetic value, which took into account their own out-of-season ovulatory activity adjusted by environmental parameters and that of their relatives. The genomic DNA of 36 ewes found regularly cycling in spring (group H) and that of 35 ewes never cycling in spring (group L) during the 2-3 yr before the present study was prepared, and the cDNA corresponding to almost all exon II was amplified and checked for the presence of Mnll restriction sites. The presence (+) or absence (-) of an Mnll site at position 605 led to genotypes "++", "+-", and "--", whose frequencies differed significantly (P < 0.001) between the H and L groups: 52.8%, 47.2%, and 0% vs. 28.5%, 42.9%, and 28.5%, respectively. Sequencing of exon II cDNA in group L ewes with genotype -- showed the presence of only one allele - with 4 mutations, while that in ewes with genotype ++ showed different types of alleles unrelated to the H or L groups. These + alleles exhibited a combination of 1 to 7 of the 8 mutations recorded in the part of exon Il studied. The genotyping of 29 ewes from the more seasonal Ile-de-France breed indicated that 38% of animals had a -- genotype and exhibited the same mutations as in the MA ewes. Finally, a comparison of I-125-melatonin binding to membrane preparations of pars tuberalis showed a lower number of binding sites (P < 0.0005) in MA ewes with genotype ++ than in those with genotype -- (43.2 +/- 4.4 vs. 75.4 +/- 8.4 fmol/mg protein in genotype ++ and genotype --, respectively). In conclusion, the data show an association between genotype -- for site Mnll at position 605 and seasonal anovulatory activity in MA ewes.

关键词：等位基因氨基酸取代/遗传学 DNA/生物合成 DNA/遗传学基因组基因型碘放射性同位素/诊断应用膜/代谢排卵/生理学垂体前叶/代谢点突变/生理学受体细胞表面/生物合成受体细胞表面/遗传学受体胞质和核/生物合成受体胞质和核/遗传学受体褪黑激素生殖/生理学逆转录聚合酶链反应季节绵羊动物女(雌)性妊娠

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The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel

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CELL 2000年第5期102卷 657-670页

作者： Minor, DL Lin, YF Mobley, BC Avelar, A Jan, YN Jan, LY Berger, JM Univ Calif San Francisco Howard Hughes Med Inst San Francisco CA 94143 USA Univ Calif San Francisco Dept Physiol San Francisco CA 94143 USA Univ Calif San Francisco Dept Biochem San Francisco CA 94143 USA Univ Calif Berkeley Dept Mol & Cell Biol Berkeley CA 94720 USA

Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Recent mutagenesis of two T1 C-terminal loop residues implicates T1 in channel gating. However, structural alterations of these mutants leave open the question concerning direct involvement of T1 in gating. We find in mammalian Kv1.2 that gating depends critically on residues at complementary T1 surfaces in an unusually polar interface. An isosteric mutation in this interface causes surprisingly little structural alteration while stabilizing the dosed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel. Together, these data suggest that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening.

关键词：氨基酸取代/遗传学结合部位圆二色性结晶学 X线电生理学静电学离子通道闸门 Kv1.2钾通道模型分子突变/遗传学卵母细胞钾/代谢钾通道/化学钾通道/遗传学钾通道/代谢钾通道电压门控蛋白质结构二级蛋白质结构三级构效关系爪蟾光滑动物

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Nonfibrillar diffuse amyloid deposition due to a γ₄₂-secretase site mutation points to an essential role for N-truncated Aβ₄₂ in Alzheimer's disease

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HUMAN MOLECULAR GENETICS 2000年第18期9卷 2589-2598页

作者： Kumar-Singh, S De Jonghe, C Cruts, M Kleinert, R Wang, R Mercken, M De Strooper, B Vanderstichele, H Löfgren, A Vanderhoeven, I Backhovens, H Vanmechelen, E Kroisel, PM Van Broeckhoven, C Univ Antwerp VIB Born Bunge Fdn Mol Genet Lab B-2610 Antwerp Belgium Graz Univ Inst Med Biol & Human Genet Lab Human Genet A-8010 Graz Austria Graz Univ Neuropathol Lab Inst Pathol A-8010 Graz Austria Rockefeller Univ Lab Mass Spectrometry New York NY 10021 USA Janssen Res Fdn B-2340 Beerse Belgium Univ Leuven Lab Neuronal Cell Biol & Gene Transfer Zwijnaarde Belgium Innogenet Zwijnaarde Belgium

Amyloidogenic processing of the amyloid precursor protein (APP) with deposition in brain of the 42 amino acid long amyloid P-peptide (A beta (42)) is considered central to Alzheimer's disease (AD) pathology. However, it is generally believed that nonfibrillar pre-amyloid A beta (42) deposits have to mature in the presence of AP(40) into fibrillar amyloid plaques to cause neurodegeneration, Here, we describe an aggressive form of AD caused by a novel missense mutation in APP (T7141) directly involving gamma -secretase cleavages of APP, The mutation had the most drastic effect on A beta (42)/A beta (40) ratio in vitro of similar to 11-fold, simultaneously increasing A beta (42) and decreasing AP(40) secretion, as measured by matrix-assisted laser disorption ionization time-of-flight mass spectrometry, This coincided in brain with deposition of abundant and predominant nonfibrillar pre-amyloid plaques composed primarily of N-truncated A beta (42) in complete absence of A beta (40) These data indicate that N-truncated A beta (42) as diffuse nonfibrillar plaques has an essential but undermined role in AD pathology, importantly, inhibiting secretion of full-length A beta (42) by therapeutic targeting of APP processing should not result in secretion of an equally toxic N-truncated A beta (42).

关键词：阿尔茨海默病/酶学阿尔茨海默病/遗传学阿尔茨海默病/代谢阿尔茨海默病/病理学氨基酸取代/遗传学淀粉样前体蛋白分泌酶类淀粉样β肽类/化学淀粉样β肽类/代谢天冬氨酸内肽酶类细胞系内肽酶类/遗传学内肽酶类/代谢酶联免疫吸附测定免疫组织化学突变误义/遗传学系谱肽碎片/化学肽碎片/代谢斑块淀粉样/化学斑块淀粉样/酶学斑块淀粉样/代谢斑块淀粉样/病理学蛋白质加工转译后溶解度光谱法质量基质辅助激光解吸电离转染女(雌)性人类男(雄)性

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The structure of the C949S mutant human α₂-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation

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JOURNAL OF STRUCTURAL BIOLOGY 2000年第1期131卷 19-26页

作者： Qazi, U Kolodziej, SJ Gettins, PGW Stoops, JK Univ Texas Sch Med Dept Pathol & Lab Med Houston TX 77030 USA Univ Illinois Dept Biochem Chicago IL 60612 USA

A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native Lu,RI structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and ru,M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity, (C) 2000 Academic Press.

关键词：氨基酸取代/遗传学结晶学 X线半胱氨酸/化学半胱氨酸/遗传学半胱氨酸/代谢内肽酶类/代谢酯类/代谢甲胺类/代谢显微镜检查电子模型分子突变/遗传学蛋白质结合蛋白质构象蛋白质折叠热力学 α巨球蛋白类/化学 α巨球蛋白类/遗传学 α巨球蛋白类/代谢 α巨球蛋白类/超微结构人类

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Attachment of coxsackievirus B3 variants to various cell lines: Mapping of phenotypic differences to capsid protein VP1

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VIROLOGY 2000年第1期275卷 77-88页

作者： Schmidtke, M Selinka, HC Heim, A Jahn, B Tonew, M Kandolf, R Stelzner, A Zell, R Univ Jena Med Ctr Inst Virol D-07745 Jena Germany Univ Tubingen Hosp Dept Mol Pathol D-72076 Tubingen Germany Med Hsch Hannover Inst Virol D-30625 Hannover Germany

The coxsackievirus as (CVB3) strain Nancy P establishes a persistent carrier-state infection without visible cytopathic effect in primary human fibroblasts (HuFi H), whereas the derivative variant PD induces a complete lysis of the cell monolayer. To define the molecular basis of this exceptional growth property the complete genomes of both viruses were sequenced and compared to all published sequences of CVB3. As a result, six unique amino acid substitutions in the VPI capsid protein were observed. Via hybrid virus construction, the lytic phenotype was transferred to a nonlytic cDNA-generated CVB3. Mapping experiments indicate that the presence of amino acid residues K78, A80, A91, and 192 in VP1 is sufficient to induce "lytic" infections in HuFi H cells. Binding assays demonstrate that CVB3 Nancy P preferentially binds to the human coxsackievirus-adenovirus receptor (CAR), while PD exhibits a very weak interaction with CAR but strong binding to the decay accelerating factor (DAF). These results suggest that the mutated amino acid residues in VPI are involved in receptor recognition/binding. Moreover, the lytic replication of CVB3 Po and the hybrid virus in various nonpermissive rodent cell lines indicates that cell surface molecules other than CAR and DAF may be involved in attachment of this variant to cell surfaces. (C) 2000 Academic Press.

关键词：氨基酸取代/遗传学抗体/药理学抗原 CD55/代谢结合部位病毒壳体/化学病毒壳体/遗传学病毒壳体/代谢细胞系细胞培养的仓鼠亚科致细胞病变病毒 DNA突变分析 DNA 重组/遗传学肠道病毒B型人/分类肠道病毒B型人/遗传学肠道病毒B型人/代谢肠道病毒B型人/致病力成纤维细胞/药物作用成纤维细胞/病理学成纤维细胞/病毒学模型分子突变/遗传学器官特异性表型多态现象遗传/遗传学蛋白质结合/药物作用蛋白质构象受体病毒/代谢病毒复制/药物作用动物人类小鼠

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