An Asymmetric Deuterium Labeling Strategy to Identify Interprotomer and Intraprotomer NOEs in Oligomeric Proteins

A major difficulty in determining the structure of an oligomeric protein by NMR is the problem of distinguishing inter- from intraprotomer NOEs. In order to address this issue in studies of the 27 kD compact trimeric domain of the MHC class II-associated invariant chain, we compared the 13C NOESY-HSQC spectrum of a uniformly 13C-labeled trimer with the spectrum of the same trimer labeled with 13C in only one protomer, and with deuterium in the other two protomers. The spectrum of the unmixed trimer included both inter- and intraprotomer NOEs while the spectrum of the mixed trimer included only intraprotomer peaks. NOEs clearly absent from the spectrum of the mixed trimer could be confidently assigned to interprotomer interactions. Asymmetrically labeled trimers were isolated by refolding a 13C-labeled shorter form of the protein with a 2H-labeled longer form, chromatographically purifying trimers with only one short chain, and then processing with trypsin to yield only protomers with the desired N- and C-termini. In contrast to earlier studies, in which statistical mixtures of differently labeled protomers were analyzed, our procedure generated only a well-defined 1:2 oligomer, and no other mixed oligomers were present. This increased the maximum possible concentration of NMR-active protomers and thus the sensitivity of the experiments. Related methods should be applicable to many oligomeric proteins, particularly those with slow protomer exchange rates.     

Topology and Oligomerization of Mono- and Oligomeric Proteins Regulate Their Half-Lives in the Cell

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Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

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Identification of a Glycosylphosphatidylinositol-anchored Plasma Membrane Protein Interacting with the C-terminus of Auxin-binding Protein 1: A Photoaffinity Crosslinking Study

Synthetic peptides corresponding to the C-terminus of auxin-binding protein 1 (ABP1) have been shown to function as auxin agonists. To define a C-terminal receptor, photoaffinity crosslinking experiments were performed using an azido derivative of a C-terminal peptide and plasma membranes from maize (Zea mays L.). The crosslinking reaction was monitored by immunoblotting using anti-ABP1 antibodies. The crosslinked proteins were isolated by 2D gel electrophoresis and identified by mass spectrometric analysis. Further, the noncrosslinked forms of these proteins were also identified. Two proteins with apparent molecular masses of 73 kDa (termed C-terminal peptide-binding protein 1, CBP1) and 35 kDa (CBP2) were specifically linked with the C-terminal peptide. CBP2 is a cytoplasmic protein that consists of two conserved domains that are characteristic of a ricin-type lectin domain. CBP2 remained in the detergent-insoluble particles and was released from the particles by the addition of monosaccharides such as methyl-β-d-galactopyranoside. CBP1 was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that CBP1 is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein. CBP1 was found to be a copper-binding protein, and is highly homologous to Arabidopsis thaliana SKU5 that contributes to directional root growth processes. Further, it is similar to A. thaliana SKS6 that contributes to cotyledon vascular patterning and to Nicotiana tabacum NTP303 that contributes to pollen tube growth. The present results indicate that ABP1 may contribute to directional cell growth processes via the GPI-anchored plasma membrane protein SKU5 and its family members.     

Proteins of the Brain Extracellular Fluid: Evidence for Release of S-100 Protein

Abstract: Extracellular protein fractions were obtained (1) by mild, isotonic irrigation of freshly perfused brain tissue; (2) by collection of proteins released into super-fusing medium by physiologically viable slices of rat hippocampus; and (3) by sampling the CSF of anesthetized rats. Analysis of the S-100 protein content of these fractions gave values of 2.8, 4.2, and 1.8 μg S-100/mg protein, respectively. These values were three- to sixfold higher than the S-100 content of the soluble cytoplasmic protein fractions from the same tissue. This several-fold higher S-100 content of the extracellular protein fractions relative to the intracellular cytoplasmic protein fractions indicates that S-100 is selectively released into the extracellular spaces of the brain. We suggest that the biological function of this CNS protein may involve intercellular transfer.     

Mannose 6-Phosphate Receptor Proteins from Reptiles and Amphibians: Evidence for the Presence of MPR 300 and MPR 46

Two mannose 6-phosphate receptors (MPR 300 and MPR 46) are involved in transport of lysosomal enzymes. Both receptors are expressed in all mammalian species studied so far and in chicken. Here we present the first report on affinity purification of both MPRs from the liver tissues of reptiles and amphibians using Sepharose divinyl sulfone phosphomannan at pH 7.0. MPR 300 from both species show similar electrophoretic mobility as mammalian MPR 300 and cross-react with an antibody directed against MPR 300 from goat liver. Furthermore, MPR 46 from reptilian liver and amphibian oocytes cross-react with peptide-specific antibodies against the cytoplasmic domain of human MPR 46 (anti-MSC1).     

Synthetic Peptide Fragments as Probes for Structure Determination of Potassium Ion-Channel Proteins

Potassium channels are a diverse class of transmembrane proteins that are responsible for diffusion of potassium ion across cell membranes. The lack of large quantities of these proteins from natural sources, is a major hindrance in their structural characterization using biophysical techniques. Synthetic peptide fragments corresponding to functionally important domains of these proteins provide an attractive approach towards characterizing the structural organization of these ion-channels. Conformational properties of peptides from three different potassium channels (Shaker, ROMK1 and minK) have been characterized in aqueous media, organic solvents and in phospholipid membranes. Techniques used for these studies include FTIR, CD and 2D-NMR spectroscopy. FTIR spectroscopy has been a particularly valuable tool for characterizing the folding of the ion-channel peptides in phospholipid membranes; the three different types of potassium channels all share a common transmembrane folding pattern that is composed of a predominantly -helical structure. There is no evidence to suggest the presence of any significant -sheet structure. These results are in excellent agreement with the crystal structure of a bacterial potassium channel (Doyle, D. A. et al. (1998) Science 280:69–77), and suggest that all potassium channel proteins may share a common folding motif where the ion-channel structure is constructed entirely from -helices.     

A Two-Plasmid System for Independent Genetic Manipulation of Subunits of Homodimeric Proteins and Selective Isolation of Chimeric Dimers

We have designed and tested a modular two-plasmid expression system which allows coexpression of two different subunits of recombinant dimeric protein in Escherichia coli and selective purification of heterodimers. We have constructed a new expression vector, pBIOEx, with p15a replication origin which allows its stable coexistence with different ColE1 group plasmids. The expression cassette of this plasmid under control of the T7 promoter contains cloning site, followed by a short sequence coding for the C-terminal extension of the recombinant protein which is a target of the in vivo biotinylation by BirA protein. The expression unit is bicistronic, the second expressed protein being BirA. We have used this plasmid together with pET30a to clone kinesin heavy-chain fragment and coexpressed the two polypeptide chains differing by tags on their C-termini and we purified heterodimers made of two recombinant molecules. The heterodimeric protein had a normal biochemical activity. There was no discrimination against heterodimer formation at the dimerization step. The system is a powerful tool in studies of different aspects of interactions between subunits of the homodimeric proteins since it makes possible separate genetic manipulations on each subunit of the dimer.     

Proteins as a potential nitrogen storage compound in bark and leaves of several softwoods

Summary The occurrence of vegetative storage proteins in the leaf and bark tissues of several softwood species during overwintering was investigated by sodium dodecyl sulphate polyacrylamide electrophoresis. Monthly protein profiles from leaves and bark of six evergreen softwood species (Pinus strobus, P. sylvestris, Picea abies, P. glauca, Abies balsamea, and Thuja occidentalis) and the bark of one deciduous softwood species (Larix decidua) suggest that storage proteins are present in bark tissues of L. decidua, Pinus sylvestris, and P. strobus. The remaining species did not show similar specific proteins. However, the total soluble protein content which was determined during active growth and during overwintering in the same tissues indicated that protein levels were higher in the winter compared to the summer in the bark of all species and in the leaves of Pinus spp. and T. occidentalis. While vegetative storage proteins do not appear prevalent in all softwood species, proteins may constitute a major form of overwintering nitrogen storage for many species.     

Development of a bifunctional crosslinking agent with potential for the preparation of immunotoxins

A new protein crosslinking agent, 2,3-dibromopropionyl-N-hydroxysuccinimide ester, has been synthesized and characterized. The potential use of this compound as a temperature-controllable heterobifunctional crosslinking agent has been investigated using model systems and its reactivity compared with that of chlorambucil-N-hydroxysuccinimide ester. The coupling of¹⁴C-labeled phenylethylamine to lysozyme has been used to illustrate the feasibility of the use of this crosslinking agent for the synthesis of immunotoxins.     

利用农杆菌介导的瞬时表达系统研究CYC类TCP蛋白的亚细胞定位

CYCLOIDEA（CYC）类TCP基因在豆科与玄参科植物中都参与了两侧对称花型的发育,但它们的具体功能有明显的差异。通过在豌豆花瓣中建立农杆菌EHA105介导的瞬时表达系统,观察到豆科植物百脉根和玄参科植物金鱼草的CYC类TCP蛋白亚细胞定位有明显区别。     

针对DNA长片段靶标的锌指筛选体系

锌指蛋白能够特异性识别目标DNA序列,常被作为分子靶向因子用于定点核酸编辑以及定点转录调控等方面,具有十分广泛的应用前景.然而,目前常规采用的实验方法得到的锌指蛋白通常结合的目标DNA序列较短,结合能力不强,因而一直难以高效运用在核酸序列与调控蛋白在基因组上的原位互作等方面的研究中.为了解决这个问题,本研究构建了一个高通量筛选系统,利用N4噬菌体gp8毒蛋白和LacZ作为报告基因,以300 bp以上的DNA长片段为靶标,来筛选能够结合多位点的锌指蛋白组合,提高锌指蛋白应用的精确度以及效率.该系统针对小鼠Nrxn-1?启动子区域进行了锌指蛋白文库筛选,得到了具有序列选择特异性的混合锌指蛋白库,并对筛选结果进行了初步功能验证.研究表明,本系统具有简便快捷的特点,不仅大幅度缩短了筛选时间,而且减少了因靶标序列DNA片段较长而不得不反复设计多位点结合锌指蛋白造成的成本浪费;筛选得到的锌指蛋白库具有较高的长片段DNA靶标结合能力和一定的序列特异性,并且能够在真核细胞内特异地结合目标DNA序列.因此,本研究建立的新型锌指筛选系统不仅可以广泛应用于高通量筛选,而且在DNA-蛋白相互作用的研究中也具有重要意义.     

Fractionation techniques in a hydro-organic environment. I. Sulfolane as a solvent for hydrophobic proteins

Sulfolane (thiophene, tethrahydro-1,1-dioxide), at concentrations of 4 M or above, is an efficient solubilizing agent for water-insoluble proteins (e.g., zein or globin chains). In comparison with urea, it appears indefinitely stable in aqueous solutions and does not chemically modify proteins upon storage. Moreover, it favors protein structure, i.e., it increases their alpha-helix content, while urea decreases it. Sulfolane is compatible with electrophoretic techniques (it only slightly reduces polyacrylamide polymerization efficiency and it does not interfere with protein and peptide detection methods) and with chromatographic methods (it has negligible A280 nm). With hydrophilic proteins, sulfolane behaves as a mild denaturant and precipitates them at concentrations between 5 and 7 M.     

A Comparison of Ypet and Firefly Luciferase as Reporter Proteins for High-Throughput Screening

When Ypet was used as a reporter protein for high-throughput screening (HTS), it showed peak fold induction and a dynamic range similar to those for firefly luciferase. We also determined that conducting a reading immediately after media aspiration was the best method for HTS. We conclude that Ypet can serve as a substitute for luciferase as a reporter protein in HTS assays.     

Design of Chimeric Proteins on the Basis of a Pentameric Superhelical Fragment of Human Cartilage Oligomeric Matrix Protein: I. The Properties of a Hybrid Containing the Immunodominant Domain of the Circumsporozoite Protein of Plasmodium falciparum

The oligomeric recombinant protein DB-1 containing the immunodominant sites of the circumsporozoite protein of Plasmodium falciparum and tetanus toxin was constructed to optimize the schemes of presentation of B-cell epitopes during vaccination with chimeric proteins without the use of adjuvants. A fragment of the pentameric coiled-coil human cartilage oligomeric matrix protein was used as an oligomerization matrix. The expression of the protein in Escherichia coli cells was studied, a method for its purification was developed, and it was biochemically characterized.     

Proteins adsorbed to a hydrophobic surface used for determination of proteolytic activity

Wettability of a thin layer of protein adsorbed to a hydrophobic surface is reduced after proteolytic digestion. Reduced wettability is demonstrated by condensation of water vapour on the surface. The condensation patterns of enzyme-treated and untreated protein layers give different light-scattering properties which can be observed by the naked eye. Based on these principles, a new simple and inexpensive method, thin layer enzyme assay (TEA), for determination of proteolytic activity, was developed. Fibrinogen, gammaglobulin (IgG), bovine serum albumin (BSA), haemoglobin, ovalbumin and gelatin were used as substrates. The proteolytic activity in 1 ng trypsin (EC 3.4.21.4) and in 1 ng pronase (EC 3.4.24.4) was reproducibly detected.     

An Oligomeric Equilibrium Intermediate as the Precursory Nucleus of Globular and Fibrillar Supramacromolecular Assemblies in a PDZ Domain

The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60-70°C small oligomers populate, showing a typical β-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of ∼12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of ∼50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60°C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ × mol⁻¹. Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions.     

Protein design as a challenge for peptide chemists

All efforts to turn the ultimate goal in protein de novo design into reality–the construction of new macromolecules with predetermined three-dimensional structure and well-defined functionality–failed because the mechanism of folding has still to be unravelled. In the present review, various attempts to apply synthetic tools for inducing native-like structural features in peptides in order to bypass the folding problem are described. Besides well-established methods for the nucleation and stabilization of secondary structures, e.g. α-helices, β-sheets and β-turns, topological templates as ‘built-in’ folding devices have more recently become the key elements for the induction of protein-like folding units (template-assembled synthetic proteins, TASP). Progress in the synthetic strategy and structural characterization of this new type of macromolecules opens the way for the design of functional TASP molecules.     

Similarity of Domain Organization of Proteins in Phylogenetically Distant Organisms as a Basis of Meiosis Conservatism

The cytological mechanism of meiosis is very conservative in all eukaryotes. Some meiosis-specific structural proteins of yeast, nematode Caenorhabditis elegans, Drosophila, and mammals, which play identical roles in cells during meiosis, do not have homology of the primary structure, but their domain organization and conformation are similar. The enzymes of meiotic recombination in yeast and plants have similar epitopes. These facts suggest that the similarity of the higher level of organization of the meiosis-specific proteins allows these proteins to form similar subcellular structures and produce similar cytological picture of meiosis and similar functions of these subcellular structures. Finally, this leads to a conservative scheme of meiosis in evolutionally distant eukaryotes.     

The use of solid waste of a nylon-6 plant as a nutrient for bacterial decolourisation of dyes

Three caprolactam-degrading bacterial isolates grew in liquid synthetic medium containing solubilised solid waste of a nylon-6 production plant as the sole source of carbon and nitrogen. Typically, the caprolactam content of solid waste was decreased by 95% in 72 h by Alcaligenes faecalis. A. faecalis was the most potent caprolactam-degrading bacterium out of the three isolates. The biomass of the bacteria obtained by growth in the solubilised solid waste medium had the ability to decolourise some synthetic azo and triphenylmethane dyes. Decolourisation of dyes was obtained in static condition, in synthetic medium which contained only the components of the solid waste as the sole sources of carbon and nitrogen and also in nutritionally rich medium. The supplementation of yeast extract to solid waste medium did not increase the efficiency of decolourisation in case of two of the bacterial cultures. Depending on the dye, medium and bacteria used, decolourisation in the range of 35–94% was achieved in 48–96 h. The decolourisation was not due to the adsorption of the dyes by the bacterial biomass except in case of Procion Blue MR and Black B. Based on these observations, the simultaneous biological treatment of the solid waste of nylon-6 plant and the decolourisation of synthetic dyes present in wastewater or solid waste is envisaged.