Cytotoxicity of a hybrid prepared by coupling diphtheria toxin A-chain with immunoglobulin Fab′ with N,N′-o-phenylenedimaleimide

A hybrid protein was prepared by coupling the A-chain of diphtheria toxin with the Fab′ fragment of immunoglobulin with $\text{N}\text{,}\text{N′}\text{-}\text{o}$-phenylenedimaleimide (PDM). Although in this hybrid, the two components were linked with each other with bonds which could not be reductively cleaved with 2-mercaptoethanol as in a hybrid cross-linked with a disulfide bond (e.g. Fab′-S-S-A-chain), it exhibited a potent cytotoxicity $\text{in}\text{vitro}$, one-third of that of Fab′-S-S-A-chain, against the target L1210 cells.     

Evolutionary Trace Annotation of Protein Function in the Structural Proteome

By design, structural genomics (SG) solves many structures that cannot be assigned function based on homology to known proteins. Alternative function annotation methods are therefore needed and this study focuses on function prediction with three-dimensional (3D) templates: small structural motifs built of just a few functionally critical residues. Although experimentally proven functional residues are scarce, we show here that Evolutionary Trace (ET) rankings of residue importance are sufficient to build 3D templates, match them, and then assign Gene Ontology (GO) functions in enzymes and non-enzymes alike. In a high-specificity mode, this Evolutionary Trace Annotation (ETA) method covered half (53%) of the 2384 annotated SG protein controls. Three-quarters (76%) of predictions were both correct and complete. The positive predictive value for all GO depths (all-depth PPV) was 84%, and it rose to 94% over GO depths 1-3 (depth 3 PPV). In a high-sensitivity mode, coverage rose significantly (84%), while accuracy fell moderately: 68% of predictions were both correct and complete, all-depth PPV was 75%, and depth 3 PPV was 86%. These data concur with prior mutational experiments showing that ET rank information identifies key functional determinants in proteins. In practice, ETA predicted functions in 42% of 3461 unannotated SG proteins. In 529 cases—including 280 non-enzymes and 21 for metal ion ligands—the expected accuracy is 84% at any GO depth and 94% down to GO depth 3, while for the remaining 931 the expected accuracies are 60% and 71%, respectively. Thus, local structural comparisons of evolutionarily important residues can help decipher protein functions to known reliability levels and without prior assumption on functional mechanisms. ETA is available at http://mammoth.bcm.tmc.edu/eta.     

Evidence for the location of the allosteric activation switch in the multisubunit phosphorylase kinase complex from mass spectrometric identification of chemically crosslinked peptides

Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.     

防护林阶段定向经营研究Ⅱ. 典型防护林种--农田防护林

以防护林阶段定向经营理论为基础,对典型防护林种-农田防护林的防护成熟、经营阶段、更新方式、方法进行了研究讨论：通过对东北地区农田防护林长期调查积累资料的分析,确定了乡土杨和杂交杨（Populus spp.)农田防护林和初始防护成熟龄和终止防护成熟龄分别为16-24年和自然成熟龄,第一代农田防护要的更新龄为32-36年,以树木径级离散度,防护成熟龄和更新龄为主要依据定量划分了杨树农田防护林的3个经营阶段,并重点讨论了不同更新方式下3个经营阶段的变化情况,给出了维持农田防护林成熟状态的疏透度调控技术及其相关的林木分级依据与标准,同时为实现定向经营的目标,提出了各个经营阶段内应采取的系列经营管理措施。     

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: Preparation and characterization

The dihydrolipoyl transacetylase core component of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N, N′-p-phenylenedimaleimide.In one approach, the N, N′-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found the N, N′-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues.Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.     

Removal of both Ycf48 and Psb27 in Synechocystis sp. PCC 6803 disrupts Photosystem II assembly and alters QA oxidation in the mature complex

The Photosystem II (PS II) assembly factors Psb27 and Ycf48 are transiently associated with PS II during its biogenesis and repair pathways. We investigated the function of these proteins by constructing knockout mutants in Synechocystis sp. PCC 6803. In ΔYcf48 cells, PS II electron transfer and stable oxygen evolution were perturbed. Additionally, Psb27 was required for photoautotrophic growth of cells lacking Ycf48 and assembly beyond the RC47 assembly complex in ΔYcf48:ΔPsb27 cells was impeded. Our results suggest the RC47 complex formed in ΔYcf48 cells is defective and that this deficiency is exacerbated if CP43 binds in the absence of Psb27.     

PCR依赖型方法构建高质量酵母基因突变文库

针对定向进化中利用亚克隆的方法建立酵母突变文库建库周期长、效率低、库容量低、丰度低等问题,建立了一种基于体内同源重组构建酵母整合型基因突变文库的新方法。步骤为：构建目标基因的重组表达质粒;以此为模版,设计长引物片段,PCR/易错PCR/DNA Shuffling等方法扩增得到两端带有与表达载体40~70 bp同源序列的突变基因;再利用PCR扩增得到表达载体;将扩增得到的目标基因和表达载体以一定的摩尔比混合电转化酵母,目标基因和表达载体在酵母体内同源重组成为完整的表达盒,整合入酵母基因组,获得基因突变文库。对构建的突变文库进行筛选,分别得到了酶活、蛋白表达量及热稳定性提高的突变株。该方法为完全PCR依赖型 (PDM),在体内构建表达盒,效率高,操作方便,将建库周期由2周缩短为3 d,将库容量从传统的103~104提高到105以上,库阳性率达到95％。     

Transmembrane Segments Form Tertiary Hairpins in the Folding Vestibule of the Ribosome

Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100 Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a “folding vestibule” that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved intramolecular crosslinking assay, that the helical transmembrane S3b–S4 hairpin (“paddle”) of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3–S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane.     

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N′-(1,4-phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of “upper dimers” (UD) characteristic of F-actin. Here we have used 90° light scattering, electron microscopy, and N,N′-(1,4-phenylene)dimaleimide cross-linking to reinvestigate relationships between changes in filament morphology, LD decay, and increase in the yield of UD during filament growth in a wide range of conditions influencing the rate of the nucleation reaction. The results show irregularity and instability of filaments at early stages of polymerization under all conditions used, and suggest that an earlier documented coassembling of LD with monomeric actin contributes to the initial disordering of the filaments rather than to the nucleation of polymerization. The effects of the type of G-actin-bound divalent cation (Ca²⁺/Mg²⁺), nucleotide (ATP/ADP), and polymerizing salt on the relation between changes in filament morphology and progress in G-actin-to-F-actin transformation show that ligand-dependent alterations in G-actin conformation determine not only the nucleation rate but also the kinetics of ordering of the filament structure in the elongation phase. The time courses of changes in the yield of UD suggest that filament maturation involves cooperative propagation of “proper” interprotomer contacts. Acceleration of this process by the initially bound MgATP supports the view that the filament-destabilizing conformational changes triggered by ATP hydrolysis and P_i liberation during polymerization are constrained by the intermolecular contacts established between MgATP monomers prior to ATP hydrolysis. An important role of contacts involving the DNase-I-binding loop and the C-terminus of actin is proposed.     

防护林阶段定向经营研究I.理论基础

基于多年防护林经营研究与实践,在分析防护林经营目的的基础上,提出了防护林阶段定向经营理论基础,以防护林的防护成熟为核心,将防护林经营阶段划分为成熟前期,即从栽植后形成相对稳定的幼林到初始防护成熟龄;防护成熟期,防护成熟状态持续的时期,即从初始防护成熟龄到终止防护成熟龄;更新期,林木接近自然成熟开始更新直到更新结束并形成相对稳定的幼林,防护林经营的定向是指在任何经营阶段内,一切经营技术与措施都是为了使防护林向着成熟的状态发展,即防护成熟是防护林经营的方向,是最终目的,为尽量维持防护林的成熟状态并使防护效益不间断,针对发育正常与低质衰退的林分,在各个经营阶段内采取相应的系列经营管理与改造措施。