蛍光分光光度計 SPEX FluoroMaxについて記述のある文献一覧です。

掲載年

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掲載誌

概要

1999

Thermodynamic study of phosphoglycerate kinase from Thermotoga maritima and its isolated domains: Reversible thermal unfolding monitored by differential scanning calorimetry and circular dichroism spectroscopy.

Zaiss, K.; Jaenicke, R.

Biochemistry, VOL. 38, NO. 14, 1999, PP. 4633-4639

The folding of phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima and its isolated N- and C-terminal domains (N1/2 and C1/2) was characterized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. At pH 3.0-4.0, reversible thermal denaturation of TmPGK occurred below 90 degreeC. The corresponding peaks in the partial molar heat capacity function were fitted by a four-state model, describing three well-defined unfolding transitions. Using CD spectroscopy, these are ascribed to the disruption of the domain interactions and subsequent sequential unfolding of the two domains. The isolated N-terminal domain unfolds reversibly between pH 3.0 and pH 4.0 to >90% and at pH 7.0 to about 70%. In contrast, the isolated engineered C-terminal domain only shows reversible thermal denaturation between pH 3.0 and pH 3.5. Neither N1/2 nor C1/2 obeys the simple two-state mechanism of unfolding. Instead, both unfold via a partially structured intermediate. In the case of N1/2, the intermediate exhibits native secondary structure and perturbed tertiary structure, whereas for C1/2 the intermediate could not be defined with certainty.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS/CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *AVIV 62DS SPECTROPOLARIMETER; *CARY 1/3 VARIAN UV ABSORPTION SPECTROPHOTOMETER; *CIRCULAR DICHROISM SPECTROSCOPY; *DIFFERENTIAL SCANNING CALORIMETRY; *ENZYMOLOGY; *EQUIPMENT; *ISOLATION/PURIFICATION TECHNIQUES: CB; *METHODOLOGY; *NANO-DSC CSC 5100 CALORIMETER; *PHOSPHOGLYCERATE KINASE; *PHOSPHOGLYCERATE KINASE PURIFICATION; *PHOTOMETRY: CB; *PROTEIN CONCENTRATION ANALYSIS; *PURIFICATION; *PURIFICATION METHOD; *SPECTROPHOTOMETRY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROMAX SPECTROPHOTOMETER; *THERMODYNAMICS; *THERMOTOGA MARITIMA

1999

The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle.

Bigelow, Diana J.; Chen, Baowei; Jones, Terry E.

Biochemistry, VOL. 38, NO. 45, Nov. 9, 1999, PP. 14887-14896

Cellular conditions in senescent skeletal muscle have been shown to result in the loss of conformational stability of the sarcoplasmic reticulum (SR) Ca-ATPase. To identify underlying structural features of age-modified Ca-ATPase, we have utilized the fluorescence properties of protein-bound probes to assess both local and global structure. We find conformational changes that include an age-related decrease in the apparent binding affinity to high affinity calcium sites detected by fluorescence signals in both tryptophans within nearby membrane-spanning helices and fluorescein isothiocyanate (FITC) bound distally to Lys515 within the nucleotide-binding site. In addition, a substantial (80%) age-related increase in the accessibility to soluble quenchers of fluorescence of FITC is observed without concomitant changes in bimolecular quenching constants (kq) for protein-bound IAEDANS, also within the nucleotide-binding domain, and tryptophans within the membrane. Using fluorescence resonance energy transfer to measure distances between IAEDANS and FITC across the nucleotide-binding domain, we find no significant age-related change in the mean donor-acceptor distance; however, significant increases are observed in the conformational heterogeneity of this domain, as assessed by the width at half-maximum (HW) of the distance distribution, increasing with age from 29.4 +- 0.8 ANG to 42.5 +- 1.1 ANG. Circular dichroism indicates that the average secondary structure is unaltered with age. Thus, these data suggest tertiary structural alterations in specific regions around the nucleotide-binding site rather than global conformational changes.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Muscular System (Movement and Support); *rat (Muridae) --middle age; *rat (Muridae) --old; *rat (Muridae) --strain-Fischer 344; *rat (Muridae) --young; *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates; *skeletal muscle --aged; *skeletal muscle --muscular system; *calcium-ATPase --analysis; *calcium-ATPase --nucleotide-binding site; *calcium-ATPase --sarcoplasmic reticulum; *calcium-ATPase --secondary structure; *calcium-ATPase --tertiary structure; *circular dichroism spectroscopy --analytical method; *circular dichroism spectroscopy --spectroscopic techniques: CB; *enzymatic digestion --cell disruption techniques; *enzymatic digestion --chemical method; *fluorescence emission spectroscopy --analytical method; *fluorescence emission spectroscopy --spectroscopic techniques: CB; * FluoroMax-2 fluorometer --equipment; * FluoroMax-2 fluorometer --SPEX; *Jasco J-710 spectropolarimeter --equipment; *SDS-polyacrylamide gel electrophoresis --gel electrophoresis; *SDS-polyacrylamide gel electrophoresis --separation method

1999

Synthesis and biological activity of polyethylene glycol-mouse nerve growth factor conjugate.

Saltzman, W. Mark .; Belcheva, Nadya; Mahoney, Melissa J.; Woodrow-Mumford, Kim

Bioconjugate Chemistry, VOL. 10, NO. 6, Nov.-Dec.,. 1999, PP. 932-937

Conjugation of poly(ethylene glycol) derivatives with therapeutic proteins is a promising approach for enhancing protein stability and, therefore, effectiveness. An N-hydroxysuccinimidyl ester of fluorescein-PEG 2000 was used for chemical modification of mouse nerve growth factor (mNGF), a dimeric protein with therapeutic potential for Alzheimer's disease. The mNGF-PEG2000-fluorescein conjugate was characterized by RP-HPLC, spectrofluorometry, and SDS-PAGE and was biologically active, as determined by two independent NGF-specific assays (enhancement of ChAT activity in fetal neurons and neurite outgrowth in PC12 cells). The conjugate was not detectable by a standard NGF ELISA, suggesting a fortuitous reduction in protein recognition by antibodies..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Nervous System (Neural Coordination); *Pharmacology.; *PC12 cell line (Muridae) --rat pheochromocytoma cells.; *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates.; *mouse nerve growth factor --analysis; *mouse nerve growth factor --Boehringer Mannheim; *polyethylene glycol derivatives --analysis; *polyethylene glycol derivatives --Shearwater Polymers Inc.; *polyethylene glycol-mouse nerve growth factor conjugate --analysis; *polyethylene glycol-mouse nerve growth factor conjugate --biological activity; *polyethylene glycol-mouse nerve growth factor conjugate --synthesis; *polyethylene glycol-mouse nerve growth factor conjugate --therapeutic potential.; *chemical conjugation --synthetic method; *chemical conjugation --Synthesis/Modification Techniques; *matrix-assisted laser/desorption ionization time-of-flight mass spectrometry --analytical method; *matrix-assisted laser/desorption ionization time-of-flight mass spectrometry --spectroscopic techniques: CB; *reversed-phase high performance liquid chromatography reversed-phase HPLC --chromatographic techniques; *reversed-phase high performance liquid chromatography reversed-phase HPLC --purification method; *reversed-phase HPLC system --equipment; *spectrofluorometry --analytical method.; *ELISA --detection method; *ELISA --detection/labeling techniques; * FluoroMax-2 spectrofluorometer --equipment; *SDS-polyacrylamide gel electrophoresis --gel electrophoresis; *SDS-polyacrylamide gel electrophoresis --separation method

1999

Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif.

Herrmann, Christian; Geyer, Matthias; Kalbitzer, Hans Robert; Praefcke, Gerrit J.K.; Schwemmle, Martin

Journal of Molecular Biology, VOL. 293, NO. 2, Sept. 17, 1999, PP. 321-332

hGBP1 is a GTPase with antiviral activity encoded by an interferon-activated activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Galpha. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested. These results will serve as a basis for the determination of the differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *guanylate-binding protein 1 --analysis; *guanylate-binding protein 1 --structure; *guanylate-binding protein 1 --third GTP-binding motif; *nucleotide --binding; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --imaging techniques; *fluorescence spectroscopy --spectroscopic techniques: CB; *fluorimetry --analytical method; *fluorimetry --Analysis/Characterization Techniques: CB; *isothermal titration calorimetry --analytical method; *isothermal titration calorimetry --Analysis/Characterization Techniques: CB; *mutagenesis --genetic method; *mutagenesis --molecular genetics/genetic engineering; *Bruker AMX-500 spectrometer --equipment; * Fluoromax2 fluorimeter --equipment; * Fluoromax2 fluorimeter --Spex Industries; *NMR --analytical method; *NMR --imaging techniques; *NMR --spectroscopic techniques: CB; *QuikChange site-directed mutagenesis kit --equipment; *QuikChange site-directed mutagenesis kit --Stratagene

1999

Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: Two different nucleotide states in one crystal.

Rudolph, M.; Wittinghofer, A.; Vetter, I.

Protein Science, VOL. 8, NO. 4, 1999, PP. 778-787

The 2.5 ANG crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42.GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.

DESCRIPTOR(S)- *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *BISCHOFF; *CDC42; *CHEMICAL MODIFICATION; *CRYSTALLIZATION; *CYSTEINE; *C18 REVERSED PHASE COLUMN; *EMBRYONIC STRUCTURE; *FLUORESCENCE SPECTROSCOPY; * FLUOROMAX SPECTROFLUORIMETER; *GDP; *GENE SEQUENCING METHOD; *GUANOSINE-5'-DIPHOSPHO-BETA-AMIDATE; *G12V-MUTANT; *HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; *HPLC; *HUMAN; *LABORATORY EQUIPMENT; *LIQUID CHROMATOGRAPHY; *MAGNESIUM ION; *MAR IMAGING PLATE SCANNER; *MASS SPECTROMETRY; *METHODOLOGY; *NUCLEOTIDE BINDING; *PLACENTA; *PROTEIN CONFORMATIONAL FLEXIBILITY; *PROTEIN SEQUENCING; *REPRODUCTIVE SYSTEM; *SEQUENCING TECHNIQUES; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX INSTRUMENTS S.A., INC.; *SYNTHETIC METHOD; *X-RAY ANALYSIS; *X-RAY DIFFRACTION

1999

Nonessential role for methionines in the productive association between calmodulin and the plasma membrane Ca-ATPase.

Squier, Thomas C.; Sun, Hongye; Weaver, Robert F.; Yin, Dan

Biochemistry, VOL. 38, NO. 41, Oct. 12, 1999, PP. 13654-13660

To investigate the role of hydrophobic interactions involving methionine side chains in facilitating the productive association between calmodulin (CAM) and the plasma membrane (PM) Ca-ATPase, we have substituted the polar amino acid Gln for Met at multiple positions in both the amino- and carboxyl-terminal domains of CaM. Conformationally sensitive fluorescence signals indicate that these mutations have little effect on the backbone fold of the carboxyl-terminal domain of CaM. The insertion of multiple Gln in either globular domain results in a decrease in the apparent affinity of CaM for the PM-Ca-ATPase. However, despite the multiple substitution of Gln for four methionines at positions 36, 51, 71, and 72 in the amino-terminal domain or for three methionines at positions 124, 144, and 145 in the carboxyl-terminal domain, these mutant CaMs are able to fully activate the PM-Ca-ATPase. Thus, although these CaM mutants have a decreased affinity for the CaM-binding site on the Ca-ATPase, they retain the ability to fully activate the Ca-ATPase at saturating concentrations of CaM. The role of individual methionines in modulating the affinity between the carboxyl terminus and the PM-Ca-ATPase was further investigated through the substitution of individual Met with Gln. Upon substitution of Met124 and Met144 with Gln, there is a 5- and 10-fold increase in the amount of CAM necessary to obtain half-maximal activation of the PM-Ca-ATPase, indicating that these methionine side chains participate in the high-affinity association between CAM and the PM-Ca-ATPase. However, substitution of Gln for Met145 results in no change in the apparent affinity between CaM and the PM-Ca-ATPase, indicating that in contrast to all other known CAM targets, Met145 does not participate in the interaction between CaM and the PM-Ca-ATPase. These results emphasize differences in the binding interactions between individual methionines in CaM and different target enzymes, and suggest that hydrophobic interactions between methionines in CaM and the binding site on the PM-Ca-ATPase are not necessary for enzyme activation. Calculation of the binding affinities of individual CaM domains associated with activation of the PM-Ca-ATPase suggests that mutations of methionines located in either domain of CaM can decrease the initial high-affinity association between CaM and the PM-Ca-ATPase, but have little effect upon the subsequent binding of the opposing globular domain. These results suggest that the initial associations between CaM and the CaM-binding sequence in the PM-Ca-ATPase are guided by nonspecific hydrophobic interactions involving both domains of CaM.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *calmodulin --analysis; *plasma membrane calcium-ATPase --analysis; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --fluorescence detection; *fluorescence spectroscopy --spectroscopic techniques: CB; * Fluoro Max-2 fluorescence spectroscope --equipment; * Fluoro Max-2 fluorescence spectroscope --Jobin Yvon Spex

1999

Localization of the binding site for modified Gb3 on verotoxin 1 using fluorescence analysis.

Picking, William D.; Lingwood, Clifford A.; McCann, Jameson A.; Nutikka, Anita

Biochemistry, VOL. 38, NO. 22, June 1, 1999, PP. 7177-7184

Verotoxins (VTs) from Escherichia coli elicit human vascular disease as a consequence of specific binding to globotriaosylceramide (Gb3) receptors on endothelial cell surfaces. Molecular models based on the VT1 crystal structure were used previously to investigate the structural basis for receptor recognition by VT1 and other verotoxins. Interestingly, these model-based predictions of glycolipid binding to VT1 differ somewhat from recently published structural data from cocrystals of the VT1 B-subunit (VT1B) and an analogue of the sugar moiety of Gb3. In this study, fluorescence spectroscopy was used to test model-based predictions of the location of Gb3 binding on the B-subunit pentamer of VT1. Resonance energy transfer was used to calculate the distance from a coumarin probe used to replace the acyl tail of Gb3 and the single tryptophan residue (Trp34) present within each VT1B monomer. The observed energy transfer efficiency (greater than 95%) suggests that these two moieties are approximately 13.3 ANG apart when a single distance is assumed. This distance is consistent with proposed models for the fit of Gb3 within the "cleft site" of the VT1 B-subunit. When the distances from Trp34 to the other coumarinGb3 molecules (bound to each of the four remaining monomers within the VT1B pentamer) are taken into consideration, it appears likely that the coumarin-modified Gb3 analogue used in this study associates with the previously proposed receptor binding site II of VT1. This is consistent with an observed binding preference of VT2c for coumarinGb3. To provide additional information on the association of Gb3 with the VT1 B-subunit, the influence of Gb3 glycolipid binding on the accessibility of Trp34 to different quenching agents in solution was then examined. Taken together, the data suggest that coumarin-labeled Gb3 preferentially binds to site II on VT1 in a position that is consistent with the previously described molecular models.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *globotriaosylceramide receptor Gb-3 receptor --analysis; *globotriaosylceramide receptor Gb-3 receptor --localization; *verotoxin 1; *absorption spectra --analytical method; *absorption spectra --optical analysis; *emission spectra --analytical method; *emission spectra --optical analysis; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *resonance energy transfer --analytical method; *resonance energy transfer --Analysis/Characterization Techniques: CB; *Spex FluoroMax spectrofluorometer --equipment

1999

Kinase assay based on thiophosphorylation and biotinylation

Nikiforov, Theo T. .; Jeong, Sang

Biotechniques, VOL. 27, NO. 6, Dec.,. 1999, PP. 1232-1238

Protein kinases catalyze the transfer of the gamma-phosphate group from ATP to a serine, threonine or tyrosine residue of an acceptor protein. These enzymes play an important role in signal transduction. New inhibitors for these enzymes are actively being sought. In this article, we present a novel approach for detecting the activity of protein kinases, which could be useful for the high-through-put screening of chemical libraries. The method is based on the use of ATPgammaS instead of ATP in the phosphorylation reaction. This results in the transfer of a thiophosphate group onto a fluorescein-labeled acceptor peptide substrate. The mixture is then treated with a sulfur-reactive iodoacetyl derivative of biotin, which leads to the modification of the nucleophilic sulfur of the thiophosphate group and the generation of a fluorescently labeled, biotinylated molecule. Finally, streptavidin is added to the mixture and it binds to all biotinylated molecules present. The binding of streptavidin to the thiophosphorylated and biotinylated kinase substrate can be conveniently detected by measuring the change in fluorescence polarization of the fluorescent dye attached to the peptide. The detection of kinase inhibitors is demonstrated. The method is completely homogeneous and does not require any separation steps..

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques.; *biotin; *enzyme inhibitors --analysis; *enzyme inhibitors --detection; *fluorescein; *fluorescent dye; *peptides; *protein kinases --applications; *protein kinases --functions; *serine.; *ATP; *electrophoresis --analytical method; *electrophoresis --electrophoretic techniques; *fluorescence polarization measurement --analytical method; *fluorescence polarization measurement --Detection/Labeling Techniques; *kinase assays --activity assays; *kinase assays --analytical method; *kinase assays --applications.; * FluoroMax-2 --applications; * FluoroMax-2 --equipment; * FluoroMax-2 --Instruments SA; *biotinylation --analysis; *biotinylation --applications; *signal transduction; *thiophosphorylation --analysis; *thiophosphorylation --applications.

1999

Identification and dynamics of a heparin-binding site in hepatocyte growth factor.

Byrd, R. Andrew; Bottaro, Donald P.; Casas-Finet, Jose R.; Coats, R. Heath; Kaufman, Joshua D.; Rubin, Jeffrey S.; Stahl, Stephen J.; Wingfield, Paul T.; Zhou, Hongjun

Biochemistry, VOL. 38, NO. 45, Nov. 9, 1999, PP. 14793-14802

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges (Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116). In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (Kd) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The Kd of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. 15N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae) --expression vector; *Bacteria; *Eubacteria; *Microorganisms; *hepatocyte growth factor --analysis; *hepatocyte growth factor --dynamics; *hepatocyte growth factor --heparin-binding site; *hepatocyte growth factor --identification; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *proton NMR spectroscopy --analytical method; *proton NMR spectroscopy --spectroscopic techniques: CB; *NMR spectroscopy --analytical method; *NMR spectroscopy --spectroscopic techniques: CB; *Shimadzu RF5301PC spectrofluorometer --equipment; *Shimadzu RF5301PC spectrofluorometer --Shimadzu Scientific Instruments; *Spex Fluoromax-2 --equipment; *Spex Fluoromax-2 --Instruments SA; *Varian UnityPlus 500 MHz spectrometer --equipment; *Varian UnityPlus 600 MHz spectrometer --equipment

1999

Formation of fibrous aggregates from a non-native intermediate: The isolated P22 tailspike beta-helix domain.

Schuler, Benjamin; Rachel, Reinhard; Seckler, Robert

Journal of Biological Chemistry, VOL. 274, NO. 26, June 25, 1999, PP. 18589-18596

In the assembly pathway of the trimeric P22 tailspike protein, the protein conformation critical for the partitioning between productive folding and off-pathway aggregation is a monomeric folding intermediate. The central domain of tailspike, a large right-handed parallel beta-helix, is essentially structured in this species. We used the isolated beta-helix domain (Bhx), expressed with a hexahistidine tag, to investigate the mechanism of aggregation without the two terminal domains present in the complete protein. Although Bhx has been shown to fold reversibly at low ionic strength conditions, increased ionic strength induced aggregation with a maximum at urea concentrations corresponding to the midpoint of urea-induced folding transitions. According to size exclusion chromatography, aggregation appeared to proceed via a linear polymerization mechanism. Circular dichroism indicated a secondary structure content of the aggregates similar to that of the native state, but at the same time their tryptophan fluorescence was largely quenched. Microscopic analysis of the aggregates revealed a variety of morphologies; among others, fibrils with fine structure were observed that exhibited bright green birefringence if viewed under cross-polarized light after staining with Congo red. These observations, together with the effects of folding mutations on the aggregation process, indicate the involvement of a partially structured intermediate distinct from both unfolded and native Bhx.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *P22 tailspike protein --beta-helix domain; *P22 tailspike protein --fibrous aggregate formation; *P22 tailspike protein --non-native intermediate; *light scattering analysis --analytical method; *light scattering analysis --Analysis/Characterization Techniques: CB; *size exclusion chromatography --analytical method; *size exclusion chromatography --liquid chromatography; *spectrofluorometry --analytical method; *spectrofluorometry --spectroscopic techniques: CB; *ultracentrifugation --analytical method; *ultracentrifugation --centrifugation techniques: CB; *Olympus BX60 microscope --equipment; *Olympus BX60 microscope --Olympus; *Perkin-Elmer MPF3 spectrofluorometer --equipment; *Perkin-Elmer MPF3 spectrofluorometer --Perkin-Elmer; *Spex FluoroMax spectrofluorometer --equipment; *protein conformation; *protein folding; *protein-protein interactions

1999

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

Nikiforov, Theo T.; Jeong, Sang

Analytical Biochemistry, VOL. 275, NO. 2, Nov. 15, 1999, PP. 248-253

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *fluorescein --fluorescent dye; *fluorescein --label; *peptide nucleic acid probes --labeling; *peptide nucleic acids --analysis; *polylysine; *DNA --amplification; *DNA --analysis; *fluorescent polarization --detection method; *fluorescent polarization --Detection/Labeling Techniques; * FluoroMax-2 --equipment; *PCR polymerase chain reaction --DNA amplification; *PCR polymerase chain reaction --DNA amplification method

1999

Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein.

Jensen, Pia Yvonne; Bonander, Nicklas; Farver, Ole; Moller, Lisbeth Birk

Biochimica et Biophysica Acta, VOL. 1434, NO. 1, Sept. 14, 1999, PP. 103-113

We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described byK0.5, determined to be 65 muM and 19 muM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration. The regulation does not occur as an 'all-or-none' cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *copper --binding; *DNA --cloning; *Menkes disease protein --analysis; *Menkes disease protein --metal binding domain; *Menkes disease protein --purification; *Menkes disease protein --structure; *far UV circular dichroism --analytical method; *far UV circular dichroism --spectroscopic techniques: CB; *protein purification --purification method; *protein purification --Isolation/Purification Techniques: CB; *Cary4.0 spectrophotometer --equipment; *DNA cloning --cloning method; *DNA cloning --Recombinant DNA Technology; * Fluoromax spectrofluorimeter --equipment; *Jasco J720 spectrometer --equipment

1999

Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin.

Basak, Ajit K.; Bolgiano, Barbara.; Crane, Dennis T.; Jepson, Marie; Miller, Julie; Naylor, Claire E.; Titball, Richard W.

Journal of Molecular Biology, VOL. 294, NO. 3, Dec. 3,. 1999, PP. 757-770

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of alpha-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd apprxeq 175-250 muM at pH 8 assuming a 1:1 stoichiometry as measured by spectroscopic methods..

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Toxicology.; *Clostridium perfringens (Endospore-forming Gram-Positives).; *Bacteria; *Eubacteria; *Microorganisms.; *alpha-toxin --analysis; *alpha-toxin --calcium-binding site; *alpha-toxin --carboxyl-terminal domain; *alpha-toxin --characterization; *alpha-toxin --crystal structure; *alpha-toxin --sequencing; *alpha-toxin --toxin.; *circular dichroism spectroscopy --analytical method; *circular dichroism spectroscopy --spectroscopic techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *protein sequencing --sequencing method; *protein sequencing --sequencing techniques.; *Jasco J-720 spectropolarimeter --equipment; *Spex Fluoromax single photon-counting spectrofluorometer --equipment; *X-ray crystallography --analytical method; *X-ray crystallography --X-ray analysis

1999

Calnexin discriminates between protein conformational states and functions as a molecular chaperone in vitro.

Williams, David B.; Cohen-Doyle, Myrna F.; Ihara, Yoshito; Saito, Yoshiro

Molecular Cell , VOL. 4, NO. 3, Sept., 1999, PP. 331-341

Although calnexin is thought to function as a molecular chaperone for glycoproteins, a prevalent view is that it cannot distinguish between protein conformational states, binding solely through its lectin site to monoglucosylated oligosaccharides. Using purified components in vitro, calnexin effectively prevented the aggregation not only of glycoproteins bearing monoglucosylated oligosaccharides but also proteins lacking N-glycans, an effect enhanced by ATP. It also suppressed the thermal denaturation of nonglycosylated proteins and enhanced their refolding in conjunction with other cellular components. Calnexin formed stable complexes with unfolded conformers of these proteins but not with the native molecules. Therefore, in addition to being a lectin, calnexin functions as a bona fide molecular chaperone capable of interacting with polypeptide segments of folding glycoproteins.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *calnexin --analysis; *calnexin --purification; *glycoproteins; *ATP; *aggregation assay --analytical method; *aggregation assay --Analysis/Characterization Techniques: CB; *calnexin purification --purification method; *calnexin purification --Isolation/Purification Techniques: CB; *electron microscopy --microscopy method; *electron microscopy --microscopy: CB; *fluorescence spectra --analytical method; *fluorescence spectra --optical analysis; *Spex FluoroMax spectrofluorometer --equipment

1999

Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.

Hardt, Wolf-Dietrich; Bader, Benjamin; Hillenbrand, Bernhard; Mirold, Susanne; Rudolph, Markus G.; Weise, Christoph; Wittinghofer, Alfred

Journal of Biological Chemistry, VOL. 274, NO. 43, Oct. 22, 1999, PP. 30501-30509

RhoGTPases are key regulators of eukaryotic cell physiology. The bacterial enteropathogen Salmonella typhimurium modulates host cell physiology by translocating specific toxins into the cytoplasm of host cells that induce responses such as apoptotic cell death in macrophages, the production of proinflammatory cytokines, the rearrangement of the host cell actin cytoskeleton (membrane ruffling), and bacterial entry into host cells. One of the translocated toxins is SopE, which has been shown to bind to RhoGTPases of the host cell and to activate RhoGTPase signaling. SopE is sufficient to induce profuse membrane ruffling in Cos cells and to facilitate efficient bacterial internalization. We show here that SopE belongs to a novel class of bacterial toxins that modulate RhoGTPase function by transient interaction. Surface plasmon resonance measurements revealed that the kinetics of formation and dissociation of the SopEcntdotCDC42 complex are in the same order of magnitude as those describedfor complex formation of GTPases of the Ras superfamily with their cognate guanine nucleotide exchange factors (GEFs). In the presence of excess GDP, dissociation of the SopEcntdotCDC42 complex was accelerated more than 1000-fold. SopE-mediated guanine nucleotide exchange was very efficient (e.g. exchange rates almost 105-fold above the level of the uncatalyzed reaction; substrate affinity), and the kinetic constants were similar to those described for guanine nucleotide exchange mediated by CDC25 or RCC1. Far-UV CD spectroscopy revealed that SopE has a high content of alpha-helical structure, a feature also found in Dbl homology domains, Sec7-like domains, and the Ras-GEF domain of Sos. Despite the lack of any obvious sequence similarity, our data suggest that SopE may closely mimic eukaryotic GEFs.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Salmonella typhimurium (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *RhoGTPase; *SopE protein --guanosine nucleotide exchange factor; *far UV-circular dichroism spectroscopy --analytical method; *far UV-circular dichroism spectroscopy --spectroscopic techniques: CB; *filter binding assays --binding assays; *filter binding assays --characterization method; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --fluorescence detection; *protein sequencing --characterization method; *protein sequencing --sequencing techniques; * Fluoromax spectrofluorometer --equipment; * Fluoromax spectrofluorometer --Fluoromax; *Jasco J710 spectropolarimeter --equipment; *Jasco J710 spectropolarimeter --Jasco
BIOLOGICAL TAXONOMIC DESCRIPTOR(S)- Enterobacteriaceae --Bacteria; Enterobacteriaceae --Eubacteria; Enterobacteriaceae --Facultatively Anaerobic Gram-Negative Rods;

1999

Bioactive surfaces via immobilization of self-assembling polymers onto hydrophobic materials.

Bromberg, Lev; Salvati, Lawrence, Jr.

Bioconjugate Chemistry, VOL. 10, NO. 4, July-Aug., 1999, PP. 678-686

Conjugation of proteins to copolymers from poly(acrylic acid) grafted onto PEO-PPO-PEO backbone (Pluronic-PAA) following adsorption of the conjugates onto hydrophobic surfaces is reported. Insulin-Pluronic-PAA conjugates show negligible internalization of insulin into human uterine smooth muscle cells as well as enhancement of mitogenic activity. Glucose-induced release of glycated albumin complexed with a Pluronic-PAA-concanavalin conjugate and adsorbed onto polystyrene nanospheres may provide a model for a glucose-responsive protein delivery system or a heterogeneous diagnostic device.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *uterine smooth muscle cell --muscular system; *uterine smooth muscle cell --reproductive system; *insulin-Pluronic-PAA conjugate; *poly(acrylic acid); *Pluronic-PAA conjugate; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *protein-Pluronic-PAA copolymer conjugation --chemical modification; *protein-Pluronic-PAA copolymer conjugation --synthetic method; *radioactivity determination --detection method; *radioactivity determination --detection/labeling techniques; *COBRA gamma counter --equipment; *COBRA gamma counter --Packard Instrument; *SPEX FluoroMax spectrofluorometer --equipment; *Wallac 1409 DSA liquid scintillation counter --equipment; *Wallac 1409 DSA liquid scintillation counter --EG&G Wallac; *X-ray photoelectron spectroscopy --analytical method; *X-ray photoelectron spectroscopy --spectroscopic techniques: CB; *X-ray photoelectron spectroscopy --X-ray analysis; *5000 Series spectrophotometer --equipment; *5000 Series spectrophotometer --Physical Electronics; *bioactive surfaces; *hydrophobic materials; *self-assembling polymers --immobilization

1999

Binding-dependent disorder-Order transition in PKIalpha: A fluorescence anisotropy study.

Johnson, David A.; Hauer, Jennifer A.; Taylor, Susan S.

Biochemistry, VOL. 38, NO. 21, May 25, 1999, PP. 6774-6780

The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKIalpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKIalpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKIalpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKIalpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKIalpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKIalpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *cAMP-dependent protein kinase cyclic AMP-dependent protein kinase ; *protein kinase inhibitor alpha PKI alpha ; *S28C --analysis; *S28C --labeling; *S28C --protein kinase inhibitor alpha; *S28C --purification; *S59C --analysis; *S59C --labeling; *S59C --protein kinase inhibitor alpha; *S59C --purification; *V3C --analysis; *V3C --labeling; *V3C --protein kinase inhibitor alpha; *V3C --purification; *fluorescein iodoacetamide labeling --labeling method; *fluorescein iodoacetamide labeling --Detection/Labeling Techniques; *protein purification --purification method; *protein purification --Isolation/Purification Techniques: CB; *steady-state emission spectra --optical analysis; *steady-state emission spectra --Analysis/Characterization Techniques: CB; *time-resolved fluorescence anisotropy --analytical method; *time-resolved fluorescence anisotropy --Analysis/Characterization Techniques: CB; *EEY scientific nanosecond spectrofluorometer --equipment; *I. S. A. Jobin Yvon-Spex FluoroMax II spectrofluorometer --equipment

1999

A theoretical model successfully identifies features of hepatitis B virus capsid assembly

Zlotnick, Adam; Endres, Dan; Johnson, Jennifer M.; Stahl, Stephen J.; Wingfield, Paul W.

Biochemistry, VOL. 38, NO. 44, Nov. 2, 1999, PP. 14644-14652

The capsids of most spherical viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any virus, the physical chemistry of virus capsid assembly is poorly understood. We have developed general models of capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent "elongation" reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of virus assembly can be related to the assembly of any "spherical" polymer. Using light scattering and size exclusion chromatography, we observed polymerization of assembly domain dimers of hepatitis B virus (HBV) capsid protein. Empty capsids assemble at a rate that is a function of protein concentration and ionic strength. The kinetics of capsid formation were sigmoidal, where the rate of the fast phase had second-power concentration dependence. The extent of assembly had third-power concentration dependence. Simulations based on the models recapitulated the concentration dependences observed for HBV capsid assembly. These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits. On the basis of this mechanism, we suggest that HBV capsid assembly could be an important target for antiviral therapeutics.

DESCRIPTOR(S)- *Membranes (Cell Biology); *Methods and Techniques; *Models and Simulations (Computational Biology); *hepatitis B virus (Hepadnaviridae); *Animal Viruses; *Microorganisms; *Viruses; *size exclusion chromatography --analytical method; *size exclusion chromatography --liquid chromatography; *SPEX Fluoromax 2 fluorometer --equipment; *viral capsid assembly --theoretical model

1999

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes.

Mattjus, P.; Molotkovsky, J. G.; Smaby, J. M.; Brown, R. E.

Analytical Biochemistry, VOL. 268, NO. 2, 1999, PP. 297-304

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The non-polar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.

DESCRIPTOR(S)- *AMINO ACIDS; *ANALYSIS; *ANALYSIS/CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *BIOLOGICAL PROCESSES; *BOVINE; *BRAIN; *EQUIPMENT; *FLUORESCENCE ENERGY TRANSFER ASSAY; *FLUOROPHORES; *GLYCOLIPIDS; *INSTRUMENTS S. A. INC.; *LIGHT SCATTERING; *LIPID TRANSFER MONITORING; *LIPID TRANSFER PROTEIN; *LIPIDS; *METHODOLOGY; *NERVOUS SYSTEM; *PROTEIN-MEDIATED GLYCOLIPID TRANSFER; *PURIFICATION; *SPEX FLUOROMAX INSTRUMENT; *USES; *VESICLE MEMBRANES