Validation of the detergent micelle classification for membrane protein crystals and explanation of the Matthews Graph for soluble proteins

Protein crystals are of wide-spread interest because many of them allow structure analyses at atomic resolution. For soluble proteins, the packing density of such crystals is distributed according to the Matthews Graph. For integral membrane proteins, the respective graph is similar but at lower density and much broader. By visualizing the relative positions and orientations of membrane proteins in crystals, it has been suggested that the detergent micelles surrounding these proteins form sheets, filaments, or remain isolated in the crystal giving rise to three distinct packing density distributions that superimpose to form the observed broad distribution. This classification was indirect because detergent is not visible in X-ray crystallography. Given the extensive work involved in analyzing detergent structure directly by neutron diffraction, it seems unlikely that a statistically relevant number of them will be established in the near future. Therefore, the proposed classification is here scrutinized by a simulation in which an average detergent-carrying membrane protein was randomly packed to form crystals. The analysis reproduced the three types of detergent structures together with their packing density distributions and relative frequencies, which validates the previous classification. The simulation program was also run for crystals from soluble proteins using ellipsoids as reference shapes and defining a shape factor that quantifies the deviation from the nearest ellipsoid. This series reproduced and thus explained the Matthews Graph.     

Membrane potentials,electrolyte contents,cell pH,and some enzyme activities of fibroblasts

Summary The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was −10.2±0.20 mV (n=390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3′,5′-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10⁻⁵ to 10⁻⁴ M), hydrocortisone (10⁻⁷ to 10⁻⁶ M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na⁺, K⁺, and Cl⁻ concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [¹⁴C] dimethyloxazolidine-2, 4-dione, and³H₂O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activiities of Na⁺, K⁺-, HCO₃ ⁻-, and Ca⁺⁺, Mg⁺⁺-ATPases in these cultured cells were 19.0±2.1, 13.6±2.1, and 6.6±1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na⁺, K⁺, Cl⁻, and H⁺ are not distributed according to their electrochemical gradients across the cell membrane. Na⁺, Cl⁻, and H⁺ are actively transported out of the cells and K⁺ into the cells. This study was supported by Grant AM20935 from the NIAMDD, NIH, Bethesda, Maryland, and National Aeronautics & Space Administration NASA-Ames Grant NAG 2-108 and U.S. Department of Energy Contract DE-AC02-76-EV-00119. D. M. W. is the recipient of a Research Career Award (5-K6-NB-13838), NINCDS, NIH.     

草地螟滞育幼虫的蛋白和核酸含量变化

为了阐明草地螟Loxostege sticticalis 滞育的分子调控基础, 本研究应用Trizol法、 DNA和蛋白质定量试剂盒、 SDS-PAGE电泳技术分别对进入滞育1, 2, 3和4个月、 解除滞育以及非滞育草地螟老熟幼虫中的核酸含量、 总蛋白含量和组分的变化进行了测定。结果表明： 滞育不同月份幼虫的总RNA含量显著低于非滞育幼虫的总RNA含量（P＜0.05）; 解除滞育幼虫的总RNA含量显著高于滞育2, 3和4个月的幼虫, 但仍显著低于非滞育的对照组。滞育不同月份、 非滞育以及解除滞育幼虫的总DNA含量没有显著差异（P＞0.05）。RNA/DNA比值随滞育的开始而显著下降, 随着滞育的结束而显著上升。滞育不同月份的幼虫总蛋白含量显著高于非滞育幼虫的总蛋白含量（P<0.05）, 但解除滞育与非滞育幼虫的总蛋白含量无显著差异。利用SDS-PAGE电泳分析发现滞育幼虫体内存在滞育关联蛋白, 蛋白条带在24 kDa左右, 但非滞育和已经解除滞育的幼虫则没有该蛋白条带。这些结果表明, 总RNA含量的降低、 RNA/DNA比值下降、 总蛋白含量的升高, 以及24 kDa蛋白的存在是草地螟幼虫滞育的主要生理特征。     

Essential functions of iron-requiring proteins in DNA replication,repair and cell cycle control

Eukaryotic cells contain numerous iron-requiring proteins such as iron-sulfur (Fe-S) cluster proteins, hemoproteins and ribonucleotide reductases (RNRs). These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these ironrequiring proteins and is genetically associated with diseases characterized by DNA repair defects in mammals. Organisms have evolved multi-layered mechanisms to regulate iron balance to ensure genome stability and cell development. This review briefly provides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.     

Comparison of Singlet and Doublet Paramecium tetraurelia: DNA Content,Protein Content and the Cell Cycle*

SYNOPSIS Doublet Paramecium tetraurelia contain either a single macronucleus which is substantially larger than that in a singlet cell, or 2 smaller macronuclei. Doublets have approximately twice the DNA content and twice the total protein content of singlets. The cell cycle of doublets is 164% as long as that of singlets, but the relative position of the macronuclear DNA synthesis period within the cell cycle is the same as in singlets. The DNA content of doublets is regulated so that differences in the number of macronuclei do not produce corresponding changes in DNA content; bimacronucleate doublets have only 27% more DNA than unimacronucleate doublets.     

二化螟滞育幼虫的蛋白和核酸含量以及保护酶活性的变化

为了阐明二化螟Chilo supprressalis滞育幼虫的分子特征及滞育期间保护酶活性的变化规律, 本研究应用Trizol法、 总量DNA提取法和蛋白定量试剂盒, 测定了在长光周期16L∶8D和25℃下发育的非滞育老熟幼虫、 在短光周期12L∶12D 和25℃下诱导滞育51 d的幼虫（称为滞育0个月）、 滞育1, 2和3个月幼虫的核酸含量和总蛋白含量; 同时应用试剂盒测定了老熟幼虫、 滞育0, 1和2个月的二化螟幼虫5种保护酶（POD, CAT, SOD, LDH和ATP酶）的活性。结果表明： 滞育幼虫的总RNA含量显著低于非滞育的老熟幼虫（P<0.05）, 而滞育1, 2和3个月的幼虫之间没有显著差异（P≥0.05）。老熟幼虫的总DNA含量显著高于滞育幼虫（P<0.05）。老熟幼虫的RNA/DNA比值较低, 滞育幼虫的RNA/DNA比值较高, RNA/DNA比值随着滞育时间的推移呈现出先上升后下降的趋势。滞育期大于1个月的幼虫中蛋白含量均显著高于非滞育的老熟幼虫（P<0.05）, 而滞育1, 2和3个月的幼虫之间没有显著差异（P≥0.05）。二化螟幼虫体内5种保护酶活性随发育阶段不同而存在差异。滞育幼虫中POD, CAT和SOD的活性随滞育时间延长而逐渐增强, 滞育2个月幼虫中的活性最高, 而非滞育老熟幼虫中的活性最低; LDH和ATP酶的活性则相反, 非滞育老熟幼虫中的活性最高, 滞育2个月幼虫中的活性最低。这些结果说明, 总RNA和DNA含量降低、 RNA/DNA比值先升后降、 总蛋白含量升高以及保护酶活性的变化是二化螟幼虫滞育过程中的主要生理特征。     

An improved method for the determination of γ-carboxyglutamic acid in proteins,bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of γ-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of γ-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of γ-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem.117, 259–265, 1981). The application of this method to the formic acid-soluble or insoluble γ-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of γ-carboxyglutamic acid content in chicken bone with age are reported.     

Osmotic second virial cross‐coefficient measurements for binary combination of lysozyme,ovalbumin, and α‐amylase in salt solutions

Interactions measurement is a valuable tool to predict equilibrium phase separation of a desired protein in the presence of unwanted macromolecules. In this study, cross‐interactions were measured as the osmotic second virial cross‐coefficients (B₂₃) for the three binary protein systems involving lysozyme, ovalbumin, and α‐amylase in salt solutions (sodium chloride and ammonium sulfate). They were correlated with solubility for the binary protein mixtures. The cross‐interaction behavior at different salt concentrations was interpreted by either electrostatic or hydrophobic interaction forces. At low salt concentrations, the protein surface charge dominates cross‐interaction behavior as a function of pH. With added ovalbumin, the lysozyme solubility decreased linearly at low salt concentration in sodium chloride and increased at high salt concentration in ammonium sulfate. The B₂₃ value was found to be proportional to the slope of the lysozyme solubility against ovalbumin concentration and the correlation was explained by preferential interaction theory. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1203–1211, 2013     

The fluorophenylalanine sensitive and resistant tobacco cell lines,TX1 and TX 4 1. DNA contents,chromosome numbers,nuclear ultrastructures,and effects of spermidine

Summary The two tobacco cell lines TX 1 (wild type) and TX 4 (Palmer andWidholm 1975), which are respectively sensitive and resistant to pfluorophenylalanine, were studied by light and electron microscopy and scanning cytophotometry. The two cell lines differ in the following respects: TX 1 cells exhibit spherical cells, a chromatin organization like in free-grown plants ofNicotiana tabacum, and mainly diploid and haploid DNA values. TX 4 cells show large elongated (thread-like) cells, a diffuse nuclear ultrastructure, and mainly tetraploid and octoploid DNA values. Chromosome number counts indicate that dividing TX 1 cells own a karyotype close to the diploid one, while dividing TX 4 cells are around the tetraploid state. In addition, the effects of an exogenous polyamine, spermidine, on chromatin organization were investigated. While the wild type cell line, TX1, did not respond to the treatment, the mutant, fluorophenylalanine resistant cell line, TX4, exhibited reduced nuclear size and increased chromatin condensation.     

Characterization of Alpine highland pastures located at different altitudes: forage evaluation,chemical composition,in vitro digestibility,fatty acid,and terpene contents

A survey was conducted over the summer of 2013 on two alpine pastures (P1 and P5), and three mountain pastures (P2, P3, and P4). The aim was to determine the botanical composition, pastoral value (PV), variation in chemical composition, gross energy, in vitro true digestibility (IVTD), in vitro indigestible neutral detergent fiber, fatty acid (FA), and terpene contents of pastures located at different altitudes. PV is highest in alpine pastures (25.7 and 26.9, for P1 and P5, respectively). Exploitation of pastures is intensive only for P1 and P2. Pastures differ in dry matter (DM), ash, crude protein, lipid, and gross energy content. Lignin content was significantly higher in P3 (125 g/kg DM), while in other pastures the variation in its average content (from 73 to 94 g/kg DM) was limited. All pastures had IVTD higher than 725 g/kg with the exception of P3 (659 g/kg). As far as FA content is concerned, in all pastures the most abundant were α-linolenic acid (from 495 to 583 g/kg of total FA) and linoleic acid (from 150 to 222 g/kg of total FA), while palmitic acid and oleic acid significantly differ among pastures. α-Pinene, β-pinene, and p-cymene were the most abundant terpenoids.     

Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Two binding sites for Rad52 have been identified on RPA. They include the previously identified C-terminal domain (CTD) of RPA32 (residues 224-271) and the newly identified domain containing residues 169-326 of RPA70. A region on Rad52, which includes residues 218-303, binds RPA70 as well as RPA32. The N-terminal region of RPA32 does not appear to play a role in the formation of the RPA:Rad52 complex. It appears that the RPA32CTD can substitute for RPA70 in binding Rad52. Sequence homology between RPA32 and RPA70 was used to identify a putative Rad52-binding site on RPA70 that is located near DNA-binding domains A and B. Rad52 binding to RPA increases ssDNA affinity significantly. Mutations in DBD-D on RPA32 show that this domain is primarily responsible for the ssDNA binding enhancement. RPA binding to Rad52 inhibits the higher-order self-association of Rad52 rings. Implications for these results for the "hand-off" mechanism between protein-protein partners, including Rad51, in homologous recombination and single-strand annealing are discussed.     

Characterization of a novel origin recognition complex-like complex: implications for DNA recognition,cell cycle control,and locus-specific gene amplification

The origin recognition complex (ORC) plays a central role in eukaryotic DNA replication. Here we describe a unique ORC-like complex in Tetrahymena thermophila, TIF4, which bound in an ATP-dependent manner to sequences required for cell cycle-controlled replication and gene amplification (ribosomal DNA [rDNA] type I elements). TIF4's mode of DNA recognition was distinct from that of other characterized ORCs, as it bound exclusively to single-stranded DNA. In contrast to yeast ORCs, TIF4 DNA binding activity was cell cycle regulated and peaked during S phase, coincident with the redistribution of the Orc2-related subunit, p69, from the cytoplasm to the macronucleus. Origin-binding activity and nuclear p69 immunoreactivity were further regulated during development, where they distinguished replicating from nonreplicating nuclei. Both activities were lost from germ line micronuclei following the programmed arrest of micronuclear replication. Replicating macronuclei stained with Orc2 antibodies throughout development in wild-type cells but failed to do so in the amplification-defective rmm11 mutant. Collectively, these findings indicate that the regulation of TIF4 is intimately tied to the cell cycle and developmentally programmed replication cycles. They further implicate TIF4 in rDNA gene amplification. As type I elements interact with other sequence-specific single-strand breaks (in vitro and in vivo), the dynamic interplay of Orc-like (TIF4) and non-ORC-like proteins with this replication determinant may provide a novel mechanism for regulation.     

Ubiquinone system,GC contents and cellular fatty acid composition of species of the form-genusMalbranchea andCoccidioides immitis for chemotaxonomic study

Ubiquinone system, GC contents and cellular fatty acid (CFA) composition were analyzed as criteria for chemotaxonomy ofMalbranchea species andCoccidioides immitis, which are suggested to be phylogenetically related. Based on the major ubiquinone,Malbranchea spp. were divided into two groups, of which one group possessed the same major ubiquinone asC. immitis. Similar GC contents and CFA profiles were obtained for the species ofMalbranchea andC. immitis. On the basis of these criteria the relationships between the fungi are discussed.     

Sphingolipid regulation of ezrin,radixin, and moesin proteins family: Implications for cell dynamics

A key but poorly studied domain of sphingolipid functions encompasses endocytosis, exocytosis, cellular trafficking, and cell movement. Recently, the ezrin, radixin and moesin (ERM) family of proteins emerged as novel potent targets regulated by sphingolipids. ERMs are structural proteins linking the actin cytoskeleton to the plasma membrane, also forming a scaffold for signaling pathways that are used for cell proliferation, migration and invasion, and cell division. Opposing functions of the bioactive sphingolipid ceramide and sphingosine-1-phosphate (S1P), contribute to ERM regulation. S1P robustly activates whereas ceramide potently deactivates ERM via phosphorylation/dephosphorylation, respectively. This recent dimension of cytoskeletal regulation by sphingolipids opens up new avenues to target cell dynamics, and provides further understanding of some of the unexplained biological effects mediated by sphingolipids. In addition, these studies are providing novel inroads into defining basic mechanisms of regulation and action of bioactive sphingolipids. This review describes the current understanding of sphingolipid regulation of the cytoskeleton, it also describes the biologies in which ERM proteins have been involved, and finally how these two large fields have started to converge. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

RepTAGs: universal tags for isolation and labeling of proteins, for labeling live mammalian cells and for drug discovery

Methods for specific immobilization, isolation and labeling of proteins are central to the elucidation of cellular functions. Based on bacterial repressor proteins, which bind to specific target sequences in response to small molecules (macrolide and tetracycline antibiotics) or environmental parameters (temperature), we have developed a set of protein tags (RepTAGs), which enable reversible immobilization of the protein of interest on a solid support for the isolation and quantification as well as for the specific labeling of target proteins with fluorescent dyes for tracking them within a complex protein mixture. Similarly, live mammalian cells were specifically labeled with a fluorescent operator sequence bound to RepTAGs, which were directed towards the cell surface for easy discrimination between transfected and untransfected cell populations. Based on the drug-responsive RepTAG-DNA interactions, it was also possible to quantify or discover antibiotics in environmental samples or compound libraries by means of rapid, sensitive detection methods involving fluorescence polarization and bioluminescence. We believe that the universally applicable RepTAGs will become essential for the analysis and manipulation of proteins in the most diverse areas of protein chemistry and cell biology.     

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimensional structures of proteins and its implications for secondary structure prediction from amino acid sequences

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected nonhomologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for αhelix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For Β-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part II. Virus,host cell protein,DNA clearance,and antibody recovery

Anion exchange membrane adsorbers are used for contaminant removal in flow‐through polishing steps in the manufacture of biopharmaceuticals. This contribution describes the clearance of minute virus of mice, DNA, and host cell proteins by three commercially available anion‐exchange membranes: Sartobind Q, Mustang Q, and ChromaSorb. The Sartobind Q and Mustang Q products contain quaternary amine ligands; whereas, ChromaSorb contains primary amine based ligands. Performance was evaluated over a range of solution conditions: 0–200 mM NaCl, pH 6.0–9.0, and flow rates of 4–20 membrane volumes/min in the presence and absence of up to 50 mM phosphate and acetate. In addition contaminant clearance was determined in the presence and absence of 5 g/L monoclonal antibody. The quaternary amine based ligands depend mainly on Coulombic interactions for removal of negatively charged contaminants. Consequently, performance of Sartobind Q and Mustang Q was compromised at high ionic strength. Primary amine based ligands in ChromaSorb enable high capacities at high ionic strength due to the presence of secondary, hydrogen bonding interactions. However, the presence of hydrogen phosphate ions leads to reduced capacity. Monoclonal antibody recovery using primary amine based anion‐exchange ligands may be lower if significant binding occurs due to secondary interactions. The removal of a specific contaminant is affected by the level of removal of the other contaminants. The results of this study may be used to help guide selection of commercially available membrane absorbers for flow‐through polishing steps. Biotechnol. Bioeng. 2013; 110: 500–510. © 2012 Wiley Periodicals, Inc.