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Temperature dependence of DNA affinity chromatography of transcription factors

ANALYTICAL BIOCHEMISTRY 2000年第2期279卷 209-217页

作者： Jarrett, HW Univ Tennessee Dept Biochem Memphis TN 38163 USA

Oligonucleotides bound by the CAAT enhancer binding protein (C/EBP), the lactose repressor, and Gal4 were chemically coupled to cyanogen bromide-activated Sepharose and the temperature dependence of transcription factor chromatography was characterized, Each transcription factor was applied to the ap appropriate column and eluted using a salt gradient at several temperatures, Each transcription factor showed a unique behavior. As temperature was increases, less salt was required to elute C/EBP, more salt was required to elute lac repressor, while Gal4 showed a biphasic dependency with the amount of salt first decreasing between 4 and 19 degrees C and then increasing above 19 degrees C. This temperature dependence is not due to protein or DNA unfolding but rather is a property of complex formation. By loading a column, washing it at a permissive temperature, and then rapidly changing the column temperature, highly selective elution can be obtained. The thermodynamics of this temperature effect are different for the binding of specific and nonspecific DNA sequences, making chromatography at different temperatures a potentially important way of purifying transcription factors, (C) 2000 Academic Press.

关键词：细菌蛋白质类/分离和提纯碱基序列 CCAAT增强子结合蛋白类色谱法亲和/方法 DNA/遗传学 DNA引物/遗传学 DNA结合蛋白质类/分离和提纯静电学大肠杆菌蛋白质类评价研究真菌蛋白质类/分离和提纯绿色荧光蛋白质类发光蛋白质类/分离和提纯核蛋白质类/分离和提纯蛋白质结合重组融合蛋白质类/分离和提纯阻遏蛋白质类/分离和提纯酿酒酵母蛋白质类温度热力学转录因子/分离和提纯

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Use of hydrophobic interaction chromatography to separate recombinant antibody fragments from associated bacterial chaperone protein GroEL

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ANALYTICAL BIOCHEMISTRY 2000年第2期278卷 239-241页

作者： O'Connor, KC Ghatak, S Stollar, BD Tufts Univ Sch Med Sackler Sch Grad Biomed Sci Dept Biochem Boston MA 02111 USA Tufts Univ Sch Med Div Hematol Oncol Dept Med Boston MA 02111 USA

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NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第2期97卷 599-604页

作者： Yuvaniyama, P Agar, JN Cash, VL Johnson, MK Dean, DR Univ Georgia Dept Chem Athens GA 30602 USA Univ Georgia Ctr Metalloenzyme Studies Athens GA 30602 USA Virginia Polytech Inst & State Univ Dept Biochem Blacksburg VA 24061 USA

The Nifs and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. Nifs is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained with in the nitrogenase component proteins, NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+,+) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with Nifs and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of Nifs. Approximately one transient [2Fe-2S] cluster is assembled per homodimer, The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU, Because homologs to both NifU and Nifs, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-s cluster-containing proteins.

关键词：氨基酸取代维涅兰德固氮菌/化学细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯铁/化学铁硫蛋白质类/化学大分子物质氧化还原分光光度法紫外线光谱分析拉曼

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MECHANISM OF REVERSIBLE GLYCINE CLEAVAGE REACTION IN ARTHROBACTER-GLOBIFORMIS - FUNCTION OF LIPOIC ACID IN CLEAVAGE AND SYNTHESIS OF GLYCINE

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1976年第1期173卷 71-81页

作者： KOCHI, H KIKUCHI, G TOHOKU UNIV SCH MEDDEPT BIOCHEMSENDAI 980JAPAN

The hydrogen carrier protein (H-protein) from Arthrobacter globiformis was found to contain one lipoic acid residue per molecule. In the glycine cleavage reaction, H-protein could be replaced by lipoic acid and in glycine synthesis, by dihydrolipoic acid. Lipoamide was less effective than lipoic acid in substituting for H-protein. In glycine synthesis with dihydrolipoic acid, even tetrahydrofolate and T-protein, an enzyme catalyzing the tetrahydrofolate-dependent step of the reaction, were not required, and the whole process of glycine synthesis could proceed in the presence of only one enzyme, P-protein, a pyridoxal phosphate-requiring enzyme. In the glycine decarboxylation reaction with lipoic acid, the decarboxylation product seemed to be bound to lipoic acid, possibly in the SCH2 NH2 form, which was extractable with benzene. The suspected intermediate compound could also be formed nonenzymically from dihydrolipoic acid, formaldehyde and ammonia, and glycine was obtained when the benzene extract containing the suspected intermediate compound was incubated with P-protein and NaHCO3 . Several lines of evidence suggest that a thiol group of dihydrolipoic acid reacts first with formaldehyde to form a hemimercaptal or mercaptal to which ammonia is subsequently fixed; the binding of ammonia, however, appears to be unstable. Based on observations with free lipoic acids, the sequence of the T-protein-dependent reaction steps in the enzymic, reversible glycine cleavage reaction is discussed.

关键词：节杆菌属/药物作用节杆菌属/代谢细菌蛋白质类/分离和提纯细菌蛋白质类/代谢细菌蛋白质类/生理学甘氨酸/代谢动力学脂类/分析脂类/药理学

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Characterisation of cellulose-binding proteins that are involved in the adhesion mechanism of Fibrobacter intestinalis DR7

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 1999年第4期51卷 491-497页

作者： Miron, J Forsberg, CW Agr Res Org Volcani Ctr Inst Anim Sci Metab Unit IL-50250 Bet Dagan Israel Univ Guelph Dept Microbiol Guelph ON N1G 2W1 Canada

Cellulose-binding proteins (CBP) isolated from cell envelopes of the cellulolytic bacterium Fibrobacter intestinalis strain DR7 were studied in order to investigate the adhesion mechanism. The proteins were examined for their reaction with antibodies that specifically block bacterial adhesion, response to glycosylation staining and monosaccharide composition. To this end, the effect of some monosaccharides (CBP components) on blocking of DR7 adhesion to cellulose was determined. Previous study had shown the occurrence of 16 CBP in the outer membrane and periplasm of DR7, of which 6 had endoglucanase activity (Miron and Forsberg 1998). Data from the present study show that most of the 16 CBP of DR7, except for the 38-, 90- and 180-kDa proteins, are glycosylated. Rabbit antibodies that specifically block DR7 adhesion were prepared by affinity preabsorption of antiserum against wild-type DR7 with bacterial cells of its adherence-defective mutant (DR7-M). The preabsorbed antibodies reacted positively in Western blotting with glycosylated CBP of 225, 200, 150, 70, 45 and <38 kDa from the DR7 outer membrane, and reacted weakly with CBP of DR7-M. Modification of glycosidic residues attached to the CBP of DR7 by periodate oxidation prevented any reaction with the preabsorbed antibodies. Monosaccharide analysis by HPLC of isolated CBP from the outer membrane and periplasm of DR7 cells, showed that galactosamine, glucosamine, galacturonic acid, and glucuronic acid were the predominant monosaccharide components of CBP that can block the adhesion of DR7 cells to cellulose. It is suggested that some glycosylated residues of CBP may have a predominant role in the adhesion of DR7 to cellulose.

关键词：细菌黏附/生理学细菌蛋白质类/化学细菌蛋白质类/分离和提纯细菌蛋白质类/代谢印迹法蛋白质纤维素/代谢色谱法高压液相电泳聚丙烯酰氨凝胶糖基化革兰氏阴性厌氧菌/代谢革兰氏阴性厌氧菌/生理学单糖类/分析动物兔

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Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第2期89卷 249-260页

作者： Martínez-Cuesta, MC Buist, G Kok, J Hauge, HH Nissen-Meyer, J Peláez, C Requena, T CSIC Inst Frio Dept Dairy Sci & Technol E-28040 Madrid Spain Univ Groningen Groningen Biomol Sci & Biotechnol Inst Dept Genet NL-9700 AB Groningen Netherlands Univ Oslo Dept Biochem N-0316 Oslo Norway

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

关键词：氨基酸序列氨基酸类/分析细菌蛋白质类/生物合成细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯细菌素类印迹法 DNA 基因细菌乳乳球菌/遗传学乳乳球菌/代谢分子序列数据多基因族肽类/化学转录遗传

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Cyclic lipoundecapeptide amphisin from Pseudomonas sp strain DSS73

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ACTA CRYSTALLOGRAPHICA SECTION C-STRUCTURAL CHEMISTRY 2001年第9期57卷 1123-1124页

作者： Sorensen, D Nielsen, TH Christophersen, C Sorensen, J Gajhede, M Univ Copenhagen Dept Chem Prot Struct Grp DK-2100 Copenhagen Denmark Univ Copenhagen Dept Chem Marine Chem Sect DK-2100 Copenhagen Denmark Royal Vet & Agr Univ Dept Ecol Sect Genet & Microbiol DK-1871 Copenhagen C Denmark

The crystal structure of the lipoundecapeptide amphisin, presented here as the tetrahydrate, C66H114N12O20. 4H(2)O, originating from non-ribosomal biosynthesis by Pseudomonas sp. strain DSS73, has been solved to a resolution of 0.65 Angstrom. The primary structure of amphisin is beta -hydroxydecanoyld-Leu-D-Asp-D-allo-Thr-D-Leu-D-Leu-D-Ser-L-Leu-D-Gln-L- Leu-l-Ile-l-Asp (Leu is leucine, Asp is aspartic acid, Thr is threonine, Ser is serine, Gln is glutamine and Ile is isoleucine). The peptide is a lactone, linking Thr4 O-gamma to the C-terminal. The stereochemistry of the beta -hydroxy acid is R. The peptide is a close analogue of the cyclic lipopeptides tensin and pholipeptin produced by Pseudomonas fluorescens. The structure of amphisin is mainly helical (3(10)-helix), with the cyclic peptide wrapping around a hydrogen-bonded water molecule. This lipopeptide is amphiphilic and has biosurfactant and antifungal properties.

关键词：细菌蛋白质类/化学细菌蛋白质类/分离和提纯结晶学 X线脂蛋白类/化学脂蛋白类/分离和提纯模型分子肽类环/化学肽类环/分离和提纯蛋白质构象假单胞菌属/化学假单胞菌属/代谢

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Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum

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SCIENCE 2000年第5493期290卷 995-997页

作者： Bath, J Wu, LJ Errington, J Wang, JC Harvard Univ Dept Mol & Cellular Biol Cambridge MA 02138 USA Univ Oxford Sir William Dunn Sch Pathol Oxford OX1 3RE England

The SpoIIIE protein of Bacillus subtilis is required for chromosome segregation during spore formation. The COOH-terminal cytoplasmic part of SpoIIIE was shown to be a DNA-dependent adenosine triphosphatase (ATPase) capable of tracking along DNA in the presence of ATP, and the NH2-terminal part of the protein was found to mediate its Localization to the division septum. Thus, during sporulation, SpoIIIE appears to act as a DNA pump that actively moves one of the replicated pair of chromosomes into the prespore. The presence of SpoIIIE homologs in a broad range of bacteria suggests that this mechanism for active transport of DNA may be widespread.

关键词：腺苷三磷酸酶类/代谢腺苷三磷酸/代谢氨基酸特征结构枯草芽孢杆菌/代谢细菌蛋白质类/化学细菌蛋白质类/分离和提纯细菌蛋白质类/代谢生物转运主动 DNA解旋酶类 DNA拓扑异构酶类 Ⅰ型/代谢 DNA 细菌/代谢 DNA 超螺旋/代谢重组蛋白质类/化学重组蛋白质类/分离和提纯重组蛋白质类/代谢 ∑因子孢子细菌/代谢转录因子

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Sulfite:Cytochrome c oxidoreductase from Thiobacillus novellus -: Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第18期275卷 13202-13212页

作者： Kappler, U Bennett, B Rethmeier, J Schwarz, G Deutzmann, R McEwan, AG Dahl, C Univ Bonn Inst Mikrobiol & Biotechnol D-53115 Bonn Germany CCLRC Daresbury Lab Warrington WA4 4AD Cheshire England Univ Bremen UFT D-28334 Bremen Germany Univ Bremen Fachbereich 2 D-28334 Bremen Germany Tech Univ Carolo Wilhelmina Braunschweig Biozentrum D-38106 Braunschweig Germany Univ Regensburg Inst Biochem D-93053 Regensburg Germany Univ Queensland Dept Microbiol & Parasitol St Lucia Qld 4072 Australia

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively;the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

关键词：氨基酸序列细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯细菌蛋白质类/代谢碱基序列细胞色素还原酶/化学细胞色素还原酶/遗传学细胞色素还原酶/分离和提纯细胞色素还原酶/代谢分子序列数据序列比对底物特异性亚硫酸脱氢酶硫杆菌属/酶学

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The essential transfer protein TraM binds to DNA as a tetramer

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第52期274卷 37421-37428页

作者： Verdino, P Keller, W Strohmaier, H Bischof, K Lindner, H Koraimann, G Karl Franzens Univ Graz Inst Phys Chem A-8010 Graz Austria Karl Franzens Univ Graz Inst Mikrobiol A-8010 Graz Austria

The TraM proteins encoded by F-like plasmids are sequence specific DNA binding proteins that are essential for conjugative DNA transfer. We investigated the quarternary structure and the DNA binding properties of the TraM wild-type protein of the resistance plasmid R1 and two mutant forms thereof. Size-exclusion chromatography and differential scanning calorimetry showed that purified TraM protein (amino acids 2-127) forms stable tetramers in solution. A truncated version of the protein termed TraMM26 (amino acids 2-56) forms dimers, Thus, the dimerization and tetramerization domains can be assigned to the N-terminal and C-terminal domains of TraM, respectively. Further analyses using chemical cross-linking and light scattering corroborated the preferentially tetrameric nature of the protein but also suggest that TraM has a tendency to form higher aggregates. Band-shift and fluorescence spectroscopy investigations of TraM-DNA complexes revealed that the TraM protein is also tetrameric when bound to its minimal DNA binding site, The deduced binding constant in the range of 10(8) M-1 demonstrated a very strong binding of TraM to its preferred DNA sequence. Secondary structure analysis based on CD measurements showed that TraM is mainly cu-helical with a significant increase in alpha-helicity (48 to 58%) upon DNA-binding, indicating an induced fit mechanism.

关键词：细菌蛋白质类/化学细菌蛋白质类/分离和提纯细菌蛋白质类/代谢圆二色性 DNA/代谢 DNA结合蛋白质类/化学蛋白质折叠蛋白质结构二级

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