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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利用转基因植物生产药用蛋白

本文简单介绍了国内外在利用转基因植物或植物病毒表达载体生产药物蛋白的研究和产业化现状及其发展趋势,并对２１世纪特别是前１０～１５年我国在该生物技术领域的研究方向、产业化重点以及产业化应注意的问题提出了相关建议。     

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

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Vital for Viruses: Intrinsically Disordered Proteins

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.     

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.     

植物生物反应器生产药物蛋白的前景

转基因植物作为生物反应器生产重要的药用蛋白,如抗体、血液替代品和疫苗,为健康保健和科学研究提供充足的生物药物,满足人们日益增长的需要。本文综述了这一领域的研究进展以及商业化前景。     

RNA结合蛋白与RNA互作鉴定技术

RNA结合蛋白（RNA binding proteins,RBPs）通过与RNA相互作用,广泛参与到RNA的剪切、转运、编辑、胞内定位及翻译调控等过程中。RNA领域尤其是非编码RNA（non-coding RNA,ncRNA）研究的快速发展,催生了多种RBPs RNAs相互作用鉴定技术。这些技术反之又推动了 RNA领域的研究进程。本文对紫外交联免疫沉淀（ultraviolet crosslinking and immunoprecipitation,CLIP）,CLIP cDNA文库高通量测序 (high-throughput sequencing of CLIP cDNA library,HITS-CLIP),光活化核苷增强的CLIP(photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation,PAR-CLIP),单核苷酸分离CLIP (individual nucleotide resolution CLIP,iCLIP),TRIBE (targets of RNA-binding protein identified by editing),RNA 标记,相互作用组捕获(interactome capture,IC) 和SerIC (serial RNA interactome capture)等RBPs-RNAs相互作用鉴定技术的基本原理和优缺点以及应用进行综述。     

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.     

Benchmarking a computational design method for the incorporation of metal ion‐binding sites at symmetric protein interfaces

The design of novel metal‐ion binding sites along symmetric axes in protein oligomers could provide new avenues for metalloenzyme design, construction of protein‐based nanomaterials and novel ion transport systems. Here, we describe a computational design method, symmetric protein recursive ion‐cofactor sampling (SyPRIS), for locating constellations of backbone positions within oligomeric protein structures that are capable of supporting desired symmetrically coordinated metal ion(s) chelated by sidechains (chelant model). Using SyPRIS on a curated benchmark set of protein structures with symmetric metal binding sites, we found high recovery of native metal coordinating rotamers: in 65 of the 67 (97.0%) cases, native rotamers featured in the best scoring model while in the remaining cases native rotamers were found within the top three scoring models. In a second test, chelant models were crossmatched against protein structures with identical cyclic symmetry. In addition to recovering all native placements, 10.4% (8939/86013) of the non‐native placements, had acceptable geometric compatibility scores. Discrimination between native and non‐native metal site placements was further enhanced upon constrained energy minimization using the Rosetta energy function. Upon sequence design of the surrounding first‐shell residues, we found further stabilization of native placements and a small but significant (1.7%) number of non‐native placement‐based sites with favorable Rosetta energies, indicating their designability in existing protein interfaces. The generality of the SyPRIS approach allows design of novel symmetric metal sites including with non‐natural amino acid sidechains, and should enable the predictive incorporation of a variety of metal‐containing cofactors at symmetric protein interfaces.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

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.     

On the Need to Develop Guidelines for Characterizing and Reporting Intrinsic Disorder in Proteins

Since the early 2000s, numerous computational tools have been created and used to predict intrinsic disorder in proteins. At present, the output from these algorithms is difficult to interpret in the absence of standards or references for comparison. There are many reasons to establish a set of standard‐based guidelines to evaluate computational protein disorder predictions. This viewpoint explores a handful of these reasons, including standardizing nomenclature to improve communication, rigor and reproducibility, and making it easier for newcomers to enter the field. An approach for reporting predicted disorder in single proteins with respect to whole proteomes is discussed. The suggestions are not intended to be formulaic; they should be viewed as a starting point to establish guidelines for interpreting and reporting computational protein disorder predictions.     

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.     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Tyrosinase catalyzed protein polymerization as an in vitro model for quinone tanning of insect cuticle

The validity of Pryor's widely accepted quinone tanning hypothesis for the sclerotization of insect cuticle was examined using an in vitro model system. Quinones generated in situ by the oxidation of catechols with mushroom tyrosinase and molecular oxygen readily reacted with test proteins such as lysozyme, ribonuclease and cytochrome-c, producing dimers, trimers, and higher oligomers. With the exception of 3,4-dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three compounds to support oligomerization of test protein was attributed to their high rate of intramolecular cyclization reaction. Radioactive incorporation studies reveal the formation of catechol-protomer adducts, as well as aryl-protein crosslinks in the reaction mixture. The above results strongly support the quinone tanning hypothesis. Based on these findings, the mechanism of cuticular sclerotization is discussed.     

Two Bacterial Small Heat Shock Proteins,IbpA and IbpB,Form a Functional Heterodimer

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other’s expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. β-hairpins formed by strands β5 and β7 of IbpA or IbpB crystallin domains associate with the other one's β-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.     

Proteins as supramolecular building blocks: Nterm‐Lsr2 as a new protein tecton

Proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics, and pharmaceuticals. The potential to produce nanomaterials from proteins is well supported by the numerous examples of self‐assembling proteins found in nature. We have explored self‐assembling proteins for use as supramolecular building blocks, or tectons, specifically the N‐terminal domain of Lsr2, Nterm‐Lsr2. A key feature of this protein is that it undergoes self‐assembly via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. Herein, we report the effects of pH and protein concentration on the oligomerization of Nterm‐Lsr2. Furthermore, via protein engineering, we have introduced a new trigger for oligomerization via enteropeptidase cleavage. The new construct of Nterm‐Lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 260–270, 2015.     

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.