1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates

Protein phosphorylation is a highly conserved mechanism for regulating protein function, being found in all prokaryotes and eukaryotes examined. Phosphorylation can alter protein activity or subcellular localization, target proteins for degradation and effect dynamic changes in protein complexes. In many cases, different kinases may be involved in each of these processes for a single protein, allowing a large degree of combinatorial regulation at the post-translational level. Therefore, knowing which kinases are activated during a response and which proteins are substrates is integral to understanding the mechanistic regulation of a wide range of biological processes. In this paper, I will describe methods for monitoring kinase activity, investigating kinase-substrate specificity, examining phosphorylation in planta and the determination of phosphorylation sites in a protein. In addition, strategic considerations for experimental design and variables will be discussed.     

中国古人类活动遗址形成过程研究的进展与思考

中国境内分布着数千处古人类活动遗址,是早期人类演化与适应行为研究的重要区域.虽然以往的研究一直关注地学手段在解读遗址地貌和地层形成过程中的作用,然而,作为正确鉴别遗址完整性和早期人类生存行为研究的遗址形成过程(或遗址成因)分析,长期以来缺少系统性的研究和关键指标的选择.本文结合古人类活动遗址保存的地貌和主要沉积物类型,...     

树木外生菌根菌与环境因子关系研究进展

土壤、气候和立地因子等环境因子影响树木外生菌根菌的种类及数量。本文通过查阅大量国内外文献 ,综述了外生菌根菌与土壤、季节气候及立地因子等环境因子的相互关系 ,探讨了环境因子对外生菌根菌的影响 ,揭示了外生菌根菌研究中所关注的环境因子问题。同时 ,提出了外生菌根菌与环境因子关系今后研究的主要内容与方向 ,旨在为森林生态系统的可持续经营和退化森林生态系统的恢复、改造等提供科学依据。     

Systematic development of computational models for the catalytic site in galactose oxidase: impact of outer-sphere residues on the geometric and electronic structures

A systematic in silico approach has been employed to generate sound, experimentally validated active-site models for galactose oxidase (GO) using a hybrid density functional, B(38HF)P86. GO displays three distinct oxidation states: oxidized [Cu(II)-Y*]; semireduced [Cu(II)-Y]; and reduced [Cu(I)-Y]. Only the [Cu(II)-Y*] and the [Cu(I)-Y] states are assumed to be involved in the catalytic cycle, but their structures have not yet been determined. We have developed several models (1-7) for the [Cu(II)-Y*] state that were evaluated by comparison of our computational results with experimental data. An extended model system (6) that includes solvent molecules and second coordination sphere residues (R330, Y405, and W290) is essential to obtain an experimentally correct electronic structure of the active site. The optimized structure of 6 resulted in a five-coordinate Cu site with a protein radical centered on the Tyr-Cys cofactor. We further validated our converged model with the largest model (7) that included additional outer-sphere residues (Q406, H334, Y329, G513, and T580) and water molecules. Adding these residues did not affect significantly the active site's electronic and geometric structures. Using both 6 and 7, we explored the redox dependence of the active-site structure. We obtained four- and three-coordinate Cu sites for [Cu(II)-Y] and [Cu(I)-Y] states, respectively, that corroborate well with the experimental data. The relative energies of these states were validated by a comparison with experimental redox potentials. Collectively, our computational GO models well reproduce the physicochemical characteristics of the individual states, including their redox behaviors.     

Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1–12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. Received: 2 May 2001 / Accepted: 3 August 2001     

两种立地条件下麻栎人工林地上部分养分的积累和分配

麻栎(Quercus acutissima)是一种分布广、用途大、耐干旱瘠薄的乔木树种, 掌握其生长规律和养分利用特性对麻栎人工林的科学经营十分必要。对安徽省滁州市红琊山林场两种立地条件下的麻栎人工林的生长和养分状况进行了对比研究, 结果表明: 在土壤含石量较高、养分含量较少的立地条件下, 12年生的麻栎人工林地上部分生物量为49 180.2 kg·hm^-2; 林木养分总累积量为633.9 kg·hm^-2, 其中N、P、K、Ca、Mg的累积量分别为119.9、18.7、88.5、368.6和38.2 kg·hm^-2。在土壤含石量较少、养分含量较高的立地条件下, 12年生麻栎人工林地上部分生物量为90 774.8 kg·hm^-2; 林木养分总累积量为993.6 kg·hm^-2, 其中N、P、K、Ca、Mg的累积量分别为203.5、23.0、146.9、553.6和66.6 kg·hm^-2。所以, 立地条件对麻栎生长和养分累积具有显著影响。较差立地条件下的麻栎对土壤养分的富集系数较大, 但其凋落物的养分含量较低。研究显示, 麻栎可以通过养分奢侈吸收、提高养分内循环、减少养分损失等途径来适应低养分环境。     

Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca(2+)-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.