モジュール型蛍光分光光度計 Fluorolog-3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

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1999

The structure and orientation of class-A amphipathic peptides on a phospholipid bilayer surface.

Clayton, A. H. A.; Sawyer, W. H.

European Biophysics Journal, VOL. 28, NO. 2, 1999, PP. 133-141

The amphipathic alpha-helix is a recognised structural motif that is shared by membrane-associating proteins and peptides of diverse function. The aim of this paper is to determine the orientation of an alpha-helical amphipathic peptide on the bilayer surface. We use five amphipathic 18-residue peptide analogues of a class A amphipathic peptide that is known to associate with a bilayer surface. Tyrosine and tryptophan are used as spectroscopic probes to sense local environments in the peptide in solution and when bound to the surface of unilamellar phosphatidylcholine vesicles. In a series of peptides, tryptophan is moved progressively along the sequence from the nonpolar face (positions 3, 7, 4) to the polar face of the peptide (positions 2, 12). The local environment of the tryptophan residue at each position is determined using fluorescence spectroscopy employing quantum yield, and the wavelength of the emission maximum as indicators of micropolarity. The exposure of the tryptophan residues at each site is assessed by acrylamide quenching. On association with vesicles, the tryptophan residues at positions 3, 7 and 14 are in nonpolar water-shielded environments, and the tryptophan at position 12 is in an exposed polar environment. The tryptophan at position 2, which is located near the bilayer-water interface, exhibits intermediate behaviour. Analysis of the second-derivative absorption spectrum confirmed that the tyrosine residue at position 7 is in a nonpolar water-shielded environment in the peptide-lipid complex. We conclude that these class A amphipathic peptides lie parallel to the lipid surface and penetrate no deeper than the ester linkages of the phospholipids.

DESCRIPTOR(S)- *ANALYTICAL METHOD; *APPLIED BIOSYSTEMS; *APPLIED BIOSYSTEMS PEPTIDE SYNTHESIZER MODEL 441A; *AVIV 62DS SPECTROMETER; *BIOCHEMISTRY AND BIOPHYSICS; *CAREY; *CAREY 5 UV-VIS SPECTROMETER; *CIRCULAR DICHROISM; *CLASS-A AMPHIPATHIC PEPTIDES; *FLUORESCENCE SPECTROSCOPY; *LABORATORY EQUIPMENT; *LIQUID CHROMATOGRAPHY; *MASS SPECTROMETRY: CB; *MATRIX-ASSISTED LASER DESORPTION AND IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY; *MEMBRANE-ASSOCIATING PROTEINS; *METHODOLOGY; *ORIENTATION; *PHOSPHATIDYLCHOLINE VESICLES; *PHOSPHOLIPID BILAYER SURFACE; *PURIFICATION METHOD; *REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; *REVERSED PHASE HPLC; *SOLID PHASE SYNTHESIS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX; *SPEX FLUOROLOG TAU-2 FREQUENCY DOMAIN FLUOROMETER; *STRUCTURE; *SYNTHESIS-MODIFICATION TECHNIQUES; *SYNTHETIC METHOD; *TRYPTOPHAN; *TYROSINE

1999

The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase.

Weber, Joachim; Senior, Alan E.; Nadanaciva, Sashi

Biochemistry, VOL. 38, NO. 24, June 15, 1999, PP. 7670-7677

beta-Arg-182 in Escherichia coli F1-ATPase (beta-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994) Nature 370, 621-628). Here we investigated the role of this residue by characterizing two mutants, betaR182Q and betaR182K. Oxidative phosphorylation and steady-state ATPase activity of purified F1 were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of beta-Trp-331 that occurred upon nucleotide binding to purified F1 from betaR182Q/betaY331W and betaR182K/betaY331W double mutants. It was found that (a) beta-Arg-182 interacts with the gamma-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) beta-Arg-182 has no functional interaction with the beta-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) beta-Arg-182 is directly involved in the stabilization of the catalytic transition state. Inthese features the role of beta-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, beta-Lys-155. A further role of beta-Arg-182 is suggested, namely involvement in conformational change at the catalytic site beta-alpha subunit interface that is required for multisite catalysis.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Escherichia coli (Enterobacteriaceae) --strain-beta R182K/beta Y331W; *Escherichia coli (Enterobacteriaceae) --strain-beta R182Q/beta Y331W; *Bacteria; *Eubacteria; *Microorganisms; *beta-arginine-182; *tryptophan --fluorescence measurement; *F-1-ATPase --analysis; *F-1-ATPase --characterization; *F-1-ATPase --purification; *spectrofluorometry --analytical method; *spectrofluorometry --fluorometry: CB; *Aminoc-Bowman 2 spectrofluorometer --equipment; *F-1-ATPase purification --purification method; *F-1-ATPase purification --Isolation/Purification Techniques: CB; * SPEX-Fluorolog 2 spectrofluorometer --equipment

1999

The glycine1 residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix

Carpenter, Katharine A.; Brown, William; Godbout, Claude; Hodzic, Lejla; Payza, Kemal; Pou, Chantevy; Roberts, Edward.; Schmidt, Ralf; Yue, Shi Yi

Biochemistry, VOL. 38, NO. 46, Nov. 16,. 1999, PP. 15295-15304

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c(D4,K8)Gal(1-16)-NH2 and c(D4,K8)Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alphaH chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c(D4,K8)Gal(1-16)-NH2 was determined to be 10.8 +- 3 ANG from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c(D4,K8)Gal(1-16)-NH2 and c(D4,K8)Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c(D4,K8)Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabililizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c(D4,K8)Gal(1-16)-NH2 and c(D4,K8)Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis..

DESCRIPTOR(S)- *Cell Biology; *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Pharmacology.; *HEK-293 cell line (Hominidae) --human embryonic kidney cells; *HEK-293 cell line (Hominidae) --transfected.; *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates.; *cyclic galanin analogs --analysis; *cyclic galanin analogs --pharmaceutical; *cyclic galanin analogs --synthesis; *galanin --agonist activity; *galanin --amino-terminal helix; *galanin --analysis; *galanin --neuropeptide.; *circular dichroism spectroscopy --analytical method; *circular dichroism spectroscopy --spectroscopic techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *reverse-phase high performance liquid chromatography --liquid chromatography; *reverse-phase high performance liquid chromatography --purification method.; *Bruker DMX-600 spectrometer --equipment; *Jasco J710 spectropolarimeter --equipment; *NMR spectroscopy --analytical method; *NMR spectroscopy --spectroscopic techniques: CB; * Spex-Fluorolog 2 spectrofluorimeter --equipment

1999

Spider hemocyanin binds ecdysone and 20-OH-ecdysone

Decker, Heinz .; Foell, Roman; Jaenicke, Elmar

Journal of Biological Chemistry, VOL. 274, NO. 48, Nov. 26,. 1999, PP. 34267-34271

Fluorescence quenching studies and binding experiments with (3H)ecdysone reveal that the respiratory protein, hemocyanin, of the tarantula Eurypelma californicum binds ecdysone. The binding constant for ecdysone ranges between 0.5 and 5 mM, indicating a low affinity binding. However, it is comparable with those found for the ecdysone binding to hexamerins from insects. Based on a comparison of sequences and x-ray structures of arthropodan hemocyanins, we propose an evolutionary conserved hydrophobic pocket in domain 1 of the hemocyanin subunit that may bind ecdysone..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques.; *Eurypelma californicum spider (Arachnida).; *Animals; *Arthropods; *Chelicerates; *Invertebrates.; *ecdysone; *hexamerins; *spider hemocyanin.; *20-hydroxyl-ecdysone; *fluorescence quenching --detection method; *fluorescence quenching --Detection/Labeling Techniques; *liquid scintillation --measurement method.; *liquid scintillation --Qualitative/Quantitative Techniques; *SPEX Fluorolog II spectrometer --equipment; *SPEX Fluorolog II spectrometer --SPEX; *molecular binding

1999

spectrophotometric and phosphorimetric study of a new class of heteroaromatic ketones: the six thienyl-pyridyl ketone isomers (TPK).

Romani, A.; Biggozi, A.; Ortica, F.; Favaro, G.

Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy, VOL. 55A, NO. 1, 1999-01, PP. 25-33

The six TPK isomers were synthesized according to Hardtmann et al. ( J. Med. Chem. , 1969, 12 , 1 093) by addition of n-thienyl-lithium to n'-pyridine-carboxaldehyde followed by chromium trioxide/acetic acid oxidation of the resulting carbinole. The liquid isomers were distilled and the solids crystallized and sublimed in vacuo . HPLC preparative purification was necessary to obtain very pure compounds. The absorption spectra were obtained from a Perkin-Elmer 16 spectrophotometer and the phosphorescence measurements from a Spex Fluorolog-2 FL112. Acid-base properties were measured in acetonitrile/H 2 O solutions. Low intensity, microsecond phosphorescence emissions (quantum yield less 0. 01) were observed in the liquids.

1999

Selective staining by the fluorochrome, 5,5'-diphenyl-9-ethyl-DiOC2(3). I. Physicochemical studies of dye-dye and dye-tissue interactions.

Hermel, Horst; Moehwald, Helmuth; Schmahl, Wolfgang

Biotechnic & Histochemistry, VOL. 74, NO. 5, Sept., 1999, PP. 221-228

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC2(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein beta-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.

DESCRIPTOR(S)- *Chemistry; *Methods and Techniques; *water/alcohol --reagent; *5,5'-diphenyl-9-ethyloxacarbocyanine --fluorochrome dye; *absorption spectra --analytical method; *absorption spectra --optical analysis; *fluorescence spectra --analytical method; *fluorescence spectra --optical analysis; *spectroscopy --analytical method; *spectroscopy --photometry: CB; *SPEX Fluorolog FL 212 spectrofluorometer --equipment; *Varian Cary 4E --equipment

1999

Role of leaflet asymmetry in the permeability of model biological membranes to protons, solutes, and gases.

Zeidel, Mark L.; Hill, Warren G.; Rivers, Rickey L.

Journal of General Physiology, VOL. 114 , NO. 3, Sept., 1999, PP. 405-414

Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50degreeC. Pr3+-treatment reduced membrane fluidity at temperatures above 40degreeC (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40degreeC occurred at temperatures 1-3degreeC higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders ofmagnitude higher at 48degree than at 24degreeC. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48degree than at 24degreeC, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO2 permeation through membranes occurs by a mechanism that is not dependent on fluidity.

DESCRIPTOR(S)- *Membranes (Cell Biology); *Methods and Techniques; *gases; *lipid bilayers --analysis; *lipid bilayers --functions; *lipid bilayers --gas permeability; *lipid bilayers --proton permeability; *lipid bilayers --solute permeability; *lipid bilayers --structure; *lipids --analysis; *solutes; *fluorescence anisotropy --analytical method; *fluorescence anisotropy --Qualitative/Quantitative Techniques; *spectrometry --analytical method; *spectrometry --spectroscopic techniques: CB; *SPEX Fluorolog 1680 double spectrometer --equipment; *SPEX Fluorolog 1680 double spectrometer --SPEX; *membrane fluidity; *temperature effects

1999

Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy.

Uhlenbeck, Olke C.; DeW Horrocks, William, Jr.; Feig, Andrew L.; Panek, Mark

Chemistry & Biology (London), VOL. 6, NO. 11, Nov., 1999, PP. 801-810

Background: Divalent metal ions serve as structural as well as catalytic cofactors in the hammerhead ribozyme reaction. The natural cofactor in these reactions is Mg(II), but its spectroscopic silence makes it difficult to study. We previously showed that a single Tb(III) ion inhibits the hammerhead ribozyme by site-specific competition for a Mg(II) ion and therefore can be used as a spectroscopic probe for the Mg(II) it replaces. Results: Lanthanide luminescence spectroscopy was used to study the coordination environment around Tb(III) and Eu(III) ions bound to the structurally well-characterized site on the hammerhead ribozyme. Sensitized emission and direct excitation experiments show that a single lanthanide ion binds to the ribozyme under these conditions and that three waters of hydration are displaced from the Tb(III) upon binding the RNA. Furthermore, we show that these techniques allow the comparison of binding affinities for a series of ions to this site. The binding affinities for ions at the G5 site correlates linearly with the function Z2/r of the aqua ion (where Z is the charge and r is the radius of the ion). Conclusions: This study compares the crystallographic nature of the G5 metal-binding site with solution measurements and gives a clearer picture of the coordination environment of this ion. These results provide one of the best characterized metal-binding sites from a ribozyme, so we use this information to compare the RNA site with that of typical metalloproteins.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *europium (III) --analysis; *europium (III) --molecular properties; *europium (III) --ribozyme binding; *hammerhead ribozyme --analysis; *hammerhead ribozyme --metal binding; *hammerhead ribozyme --molecular properties; *magnesium (II) --analysis; *magnesium (II) --molecular properties; *magnesium (II) --ribozyme binding; *metal catalytic cofactors --analysis; *metal catalytic cofactors --functions; *metalloproteins --analysis; *terbium (III) --analysis; *terbium (III) --molecular properties; *terbium (III) --ribozyme binding; *RNA --analysis; *luminescence lifetime measurement --analytical method; *luminescence lifetime measurement --Detection/Labeling Techniques; *luminescence spectroscopy --analytical method; *luminescence spectroscopy --spectroscopic techniques: CB; *polyacrylamide gel electrophoresis --gel electrophoresis; *polyacrylamide gel electrophoresis --purification method; *Applied Biosystems ABI 394 automated RNA synthesized --equipment; *Applied Biosystems ABI 394 automated RNA synthesized --Applied Biosystems; *RNA synthesis --synthetic method; *RNA synthesis --Synthesis/Modification Techniques; *Spex Fluorolog fluorometer --equipment; *metal ion competition --analysis

1999

Photoluminescence and vibrational spectroscopic studies on weathered uranium oxides

Eastwood, DeLyle; Martin, Jeffrey B.; Burggraf, Larry W.; Rand, Dennis S.; Zickafoose, Matthew S.; Perry, Dale L.

Proceedings of SPIE - The International Society for Optical Engineering v 3534 1999. p 487-495
1999

Spectroscopic studies were performed both on uranium oxides as baseline and on uranium oxides artificially weathered under known laboratory conditions in air, varying humidity, carbon dioxide concentration, temperature and exposure to UV light. Spectroscopic techniques included photoluminescence and diffuse reflectance FTIR. Photoluminescence measurements were made using a Spex Fluorolog-3 spectrofluorometer with phosphorimeter. FTIR measurements were made using a Bomem MB157 FTIR spectrophotometer with DTGS detector and approximately 450 cmSUP minus SUP1 cut-off and a Graseby Selector diffuse reflectance accessory with speciaL cells and diamond dust as diluent and internal standard. Weathered-related reactions involving the uranium oxides that have been studied include oxidation and the formation of hydroxides and carbonates. Data are discussed with respect to both the reactions of the uranium oxides in the study and in context of reaction chemistry and mechanisms that have been previously documented. The results will be discussed in the context of environmental monitoring. (Author abstract) 23 Refs.

1999

Photochemistry of aromatic polyesters: Mechanism and intermediates

Hoyle, Charles E.; Ziemer, Michael D.; Rufus, Bernard; Viswanathan, Kalyanaraman; Hill, David; Hunter, David; Pomery, Peter

American Chemical Society, Polymer Priprints, Division of Polymer Chemistry v 40 n 2 1999. p 758-759, 1999

A model diaryl ester (DCHMT) was synthesized by reacting cyclohexylmethanol and terephthaloyl chloride to simulate the photochemistry and photophysics of the cyclohexyldimethanol-derived repeat unit in PCHDM polyesters. Steady-state fluorescence measurements were conducted using a SPEX Fluorolog-2 fluorimeter and time-resolved fluorescence were recorded using an Edinburgh Instruments time-correlated single-photon-counting fluorescence spectrometer. Ultraviolet spectra were recorded using a Cary 500 spectrophotometer. 11 Refs.

1999

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: Evidence for interdomain interaction.

Prendergast, Franklyn G.; Moncrieffe, Martin C.; Guzman, Georgiana; Miller, Todd E.; Potter, James D.; Venyaminov, Sergei Yu

Biochemistry, VOL. 38, NO. 37, Sept. 14, 1999, PP. 11973-11983

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved 1Lb band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Muscular System (Movement and Support); *chicken (Galliformes); *Animals; *Birds; *Chordates; *Nonhuman Vertebrates; *Vertebrates; *skeletal troponin C --analysis; *skeletal troponin C --structure; *far-UV circular dichroism spectroscopy --analytical method; *far-UV circular dichroism spectroscopy --spectroscopic techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --fluorescence detection; *fluorescence spectroscopy --spectroscopic techniques: CB; *CARY 2200 spectrophotometer --equipment; *CARY 2200 spectrophotometer --Varian; *FS900CDT spectrofluorometer --equipment; *FS900CDT spectrofluorometer --Edinburgh Instruments; *J-710 spectropolarimeter --equipment; *J-710 spectropolarimeter --JASCO; *MPF-66 spectrofluorometer --equipment; *MPF-66 spectrofluorometer --Perkin-Elmer; *Spex Fluorolog spectrofluorometer --equipment; *Spex Fluorolog spectrofluorometer --Spex

1999

New organosilicon polymer for the extraction and luminescence analysis of uranyl in environmental samples.

Campiglia, A. D.; Arruda, A. F.; Boudjouk, P.; Chauhan, B. P. S.

Analytica Chimica Acta, VOL. 396, NO. 2-3, Sept. 20, 1999, PP. 263-272

A new polymer for the extraction and luminescence analysis of uranyl in environmental samples is presented. High extraction efficiencies (above 90%) of uranyl concentrations as low as part-per-billion were obtained in water and acidic solutions. The presence of concomitants in water samples did not affect the extraction capability of the new material. The luminescence characteristics of the uranyl-polymer complex demonstrate the potential for the development of a uranium phosphorescence sensor.

DESCRIPTOR(S)- *Methods and Techniques; *Pollution Assessment Control and Management; *organosilicon polymer --extraction capability; *organosilicon polymer --sensor methodology potential; *uranyl --acidic solutions; *uranyl --analysis; *uranyl --concentrations; *uranyl --environmental; *uranyl --extraction; *uranyl --water solutions; *uranyl acetate --analysis; *uranyl acetate --Pfaltz and Bauer; *uranyl-polymer complex --analysis; *uranyl-polymer complex --luminescence characteristics; *batch equilibrium method --extraction method; *batch equilibrium method --Isolation/Purification Techniques; *model FL3-11 Fluorolog-3 spectrofluorimeter --laboratory equipment; *model FL3-11 Fluorolog-3 spectrofluorimeter --JobinYvon-Spex; *time-resolved luminescence spectroscopy --analytical method; *time-resolved luminescence spectroscopy --spectroscopic techniques:CB; *IR spectroscopy --analytical method; *IR spectroscopy --spectrophotometry: CB; *Spex 1934 pulsed-lamp phosphorimeter --laboratory equipment; *2020 Galaxy Fourier transform IR spectrometer --laboratory equipment; *2020 Galaxy Fourier transform IR spectrometer --Mattson Instruments

1999

New donor-acceptor pair for fluorescent immunoassays by energy transfer

Gauglitz, Guenter .; Brecht, Andreas; Egelhaaf, Hans-Joachim; Oelkrug, Dieter; Schobel, Uwe

Bioconjugate Chemistry, VOL. 10, NO. 6, Nov.-Dec.,. 1999, PP. 1107-1114

A novel Foerster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R0 value of 68.7 ANG is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R0 value of 85.2 ANG. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The Foerster distances could be calculated usingthe dimer absorption spectra to 83.9 and 83.6 ANG for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in Foerster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Pesticides.; *simazine --analysis; *simazine --pesticide.; *Cy5 --analysis; *Cy5 --cyanine dye; *Cy5 --dye; *Cy5 --Amersham Life Science; *Cy5.5 --analysis; *Cy5.5 --cyanine dye; *Cy5.5 --dye; *Cy5.5 --Amersham Life Science; *Forster donor-acceptor dye --analysis; *Forster donor-acceptor dye --characterization; *Forster donor-acceptor dye --dye; *Forster donor-acceptor dye --photophysical properties; *fluorescence absorption spectroscopy --analytical method; *fluorescence absorption spectroscopy --spectroscopic techniques: CB; *fluorescent immunoassay --detection method; *fluorescent immunoassay --detection/labeling techniques; *fluorescent immunoassay --resonance energy transfer.; *Perkin-Elmer 2 spectrophotometer --equipment; *Spex Fluorolog 222 spectrofluorometer --equipment

1999

New approach for screening polycyclic aromatic hydrocarbons in water samples.

Campiglia, Andres D.; Hagestuen, Erik D.

Talanta, VOL. 49, NO. 3, July, 1999, PP. 547-560

For the first time, solid-phase extraction (SPE) has been combined to room-temperature phosphorimetry (RTP) to determine the 16 polycylic aromatic hydrocarbons related as major pollutants by the US Environmental Protection Agency (EPA). These include naphthalene, anthracene, acenaphthylene, acenaphthene, fluorene, fluoranthene, benzo(a)anthracene, benzo(k)fluoranthene, benzo(b)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, pyrene, chrysene, phenanthrene, benzo(g,h,i)perylene and dibenzo(a,h)anthracene. The pre-concentration factor obtained by SPE, combined with the sensitivity of RTP, resulted in calibration curves with linear dynamic ranges at the parts-per-billion level (ng ml-1). The limits of detection were estimated at the parts-per-trillion level (pg ml-1). Several pollutants usually encountered in water samples were tested for interference. These included polychlorinated biphenyls, pesticides, and volatile organic compounds. As a result of the appropriate combinationof excitation wavelength (330 nm) and phosphorescence enhancers (0.1 M TINO3 and 0.05 M sodium dodecyl sulfate, SDS), no interference was observed. The results demonstrate the potential of SPE-RTP for screening polycyclic aromatic hydrocarbons (PAHs) in environmental waters.

DESCRIPTOR(S)- *Methods and Techniques; *Pollution Assessment Control and Management; *acenaphthene --analysis; *acenaphthene --extraction; *acenaphthene --pollutant; *acenaphthene --screening; *acenaphthene --Aldrich; *acenaphthylene --analysis; *acenaphthylene --extraction; *acenaphthylene --pollutant; *acenaphthylene --screening; *acenaphthylene --Aldrich; *anthracene --analysis; *anthracene --extraction; *anthracene --pollutant; *anthracene --screening; *anthracene --Aldrich; *benzo(a)anthracene --analysis; *benzo(a)anthracene --extraction; *benzo(a)anthracene --pollutant; *benzo(a)anthracene --screening; *benzo(a)anthracene --Aldrich; *benzo(a)pyrene --analysis; *benzo(a)pyrene --extraction; *benzo(a)pyrene --pollutant; *benzo(a)pyrene --screening; *benzo(a)pyrene --Aldrich; *benzo(b)fluoranthene --analysis; *benzo(b)fluoranthene --extraction; *benzo(b)fluoranthene --pollutant; *benzo(b)fluoranthene --screening; *benzo(b)fluoranthene --Aldrich; *benzo(g,h,i)perylene --analysis; *benzo(g,h,i)perylene --extraction; *benzo(g,h,i)perylene --pollutant; *benzo(g,h,i)perylene --screening; *benzo(g,h,i)perylene --Aldrich; *benzo(k)fluoranthene --analysis; *benzo(k)fluoranthene --extraction; *benzo(k)fluoranthene --pollutant; *benzo(k)fluoranthene --screening; *benzo(k)fluoranthene --Aldrich; *chrysene --analysis; *chrysene --extraction; *chrysene --pollutant; *chrysene --screening; *chrysene --Aldrich; *dibenzo(a,h)anthracene --analysis; *dibenzo(a,h)anthracene --extraction; *dibenzo(a,h)anthracene --pollutant; *dibenzo(a,h)anthracene --screening; *dibenzo(a,h)anthracene --Aldrich; *fluoranthene --analysis; *fluoranthene --extraction; *fluoranthene --pollutant; *fluoranthene --screening; *fluoranthene --Aldrich; *fluorene --analysis; *fluorene --extraction; *fluorene --pollutant; *fluorene --screening; *fluorene --Aldrich; *indeno(1,2,3-cd)pyrene --analysis; *indeno(1,2,3-cd)pyrene --extraction; *indeno(1,2,3-cd)pyrene --pollutant; *indeno(1,2,3-cd)pyrene --screening; *indeno(1,2,3-cd)pyrene --Chem Service; *naphthalene --analysis; *naphthalene --extraction; *naphthalene --pollutant; *naphthalene --screening; *naphthalene --Aldrich; *phenanthrene --analysis; *phenanthrene --extraction; *phenanthrene --pollutant; *phenanthrene --screening; *phenanthrene --Aldrich; *polycyclic aromatic hydrocarbons --analysis; *polycyclic aromatic hydrocarbons --extraction; *polycyclic aromatic hydrocarbons --pollutant; *polycyclic aromatic hydrocarbons --screening; *polycyclic aromatic hydrocarbons --Aldrich; *polycyclic aromatic hydrocarbons --Chem Service; *pyrene --analysis; *pyrene --extraction; *pyrene --pollutant; *pyrene --screening; *pyrene --Aldrich; *model FL3-11 fluorolog-3 spectrofluorimeter --laboratory equipment; *model FL3-11 fluorolog-3 spectrofluorimeter --Jobin Yvon-Spex; *room-temperature phosphorimetry --analytical method; *room-temperature phosphorimetry --photometry: CB; *solid phase extraction --extraction method; *solid phase extraction --Isolation/Purification Techniques: CB; *water

1999

Molecular mechanisms of peptide loading by the tumor rejection antigen/heat shock chaperone gp96 (GRP94).

Sastry, Srin; Linderoth, Nora

Journal of Biological Chemistry, VOL. 274, NO. 17, April 23, 1999, PP. 12023-12035

Complexes of gp96/GRP94 and peptides have been shown to elicit immunogenicity. We used fluorescence to understand peptide association with gp96. A pyrenepeptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low salt (20 mM NaCl), the peptide binds gp96 with a strong affinity (apprx100-150 nM) and forms pyrene excimers, suggesting that the peptides were assembled as dimers. In high salt (2.2 M NaCl), although peptide binding was stronger (Ka apprxeq 55 nM) than in low salt, pyrene excimers were absent, implying that peptides were father apart in the complex. Heat shock-activated peptide binding exhibited characteristics of both low salt and high salt modes of binding. Anisotropy and average lifetime of the bound pyrene suggested that peptides were probably located in a solvent-accessible hydrophobic binding pocket in low salt, whereas in high salt, the peptide may be buried in a less hydrophobic (more hydrophilic) environment. These results suggested that peptide-gp96 complexes were assembled in several different ways, depending on the assembly conditions. Resonance energy transfer between the intrinsic tryptophan(s) in gp96 and pyrene suggested that one or more tryptophan residues were within the critical Forster distance of 27-30 ANG from the pyrene in the bound peptide. It is proposed that peptides are assembled within higher order gp96 complexes (dimers, etc.) in a hydrophobic pocket and that there may be a conformational change in gp96 leading to an open configuration for peptide loading.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *gp96 protein GRP94 protein --analysis; *gp96 protein GRP94 protein --heat shock chaperone; *gp96 protein GRP94 protein --tumor rejection antigen; *dynamic light scattering --analytical method; *dynamic light scattering --Analysis/Characterization Techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *DynaPro light scattering photon counting instrument --laboratory equipment; *DynaPro light scattering photon counting instrument --ProteinSolutions; *JOBIN YVON/SPEX fluorolog3 instrument --laboratory equipment; *JOBIN YVON/SPEX fluorolog3 instrument --Instruments; *JOBIN YVON/SPEX fluorolog3 instrument --S.A. Inc.; *peptide loading --molecular mechanisms

1999

Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: An amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein.

Matthews, C. Robert; Zitzewitz, Jill A.

Biochemistry , VOL. 38, NO. 31, Aug. 3, 1999, PP. 10205-10214

The alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli is a 268-residue 8-stranded beta/alpha barrel protein. Two autonomous folding units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion (Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835). The larger, amino-terminal fragment, alphaTS(1-188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near-and far-UV circular dichroism spectroscopies. The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 +- 0.19 kcal mol-1 in the absence of denaturant and a corresponding m value of 1.07 +- 0.05 kcal mol-1 M-1. Similar to the full-length protein, the unfolding of the fragment shows two kinetic phases which arise from the presence of two discrete native state populations. Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length alphaTS. Refolding of alphaTS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of alphaTS(1-188) appears to be a simplified version of the mechanism for the full-length protein (Bilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochemistry 38, 1018-1029). Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment. The off- and on-pathway intermediates that exist for both full-length alphaTS and alphaTS(1-188) may reflect the preponderance of local interactions in the beta/alpha barrel motif.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *tryptophan synthase --alpha-subunit; *tryptophan synthase --amino-terminal autonomous folding unit; *tryptophan synthase --analysis; *tryptophan synthase --folding kinetics; *absorbance spectroscopy --analytical method; *absorbance spectroscopy --spectroscopic techniques: CB; *equilibrium centrifugation --centrifugation techniques: CB; *equilibrium centrifugation --separation method; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *protein purification --purification method; *protein purification --Isolation/Purification Techniques: CB; *site-directed mutagenesis --genetic method; *site-directed mutagenesis --mutagenesis; *site-directed mutagenesis --protein engineering; *Aviv CARY 118DS spectrometer --equipment; *Aviv Model 202SF CD spectrophotometer --equipment; *Aviv Model 62DS CD spectrophotometer --equipment; *Beckman XL-I ultracentrifuge --equipment; *Spex Fluorolog 2 spectrometer --equipment; *UV circular dichroism spectroscopy --analytical method; *UV circular dichroism spectroscopy --spectroscopic techniques: CB

1999

Modification of liposomes with N-substituted polyacrylamides: Identification of proteins adsorbed from plasma.

Brash, J. L.; Cornelius, R. M.; Winnik, F. M.; Yamazaki, A.

Biochimica et Biophysica Acta, VOL. 1421, NO. 1, Sept. 21, 1999, PP. 103-115

Liposomes prepared from DMPC (80%) and cholesterol (20%) were modified with a series of hydrophobically modified N-substituted polyacrylamides, namely, poly(N-isopropylacrylamide) (PNIPAM), poly(N,N-bis(2-methoxyethyl) acrylamide) (PMEAM), and poly((3-methoxypropyl)acrylamide) (PMPAM). The hydrophobic group, N-(4-(1-pyrenylbutyl)-N-n-octadecylamine was attached to one end of the polymer chains to serve as an anchor for incorporation into the liposome bilayer. Liposome-polymer interactions were confirmed using fluorescence spectroscopy and chemical analysis. Microscopy revealed differences in aggregation tendency between unmodified and polymer-modified liposomes. Proteins adsorbed to liposome surfaces during exposure to human plasma were identified by immunoblot analysis. It was found that both unmodified and polymer-modified liposomes adsorb a wide variety of plasma proteins. Contact phase coagulation proteins, complement proteins, cell-adhesive proteins, serine protease inhibitors, plasminogen, antithrombin III, prothrombin, transferrin, alpha2-microglobulin, hemoglobin, haptoglobin and beta-lipoprotein as well as the major plasma proteins were all detected. Some differences were found between the unmodified and polymer-modified liposomes. The unmodified liposomes adsorbed plasminogen mainly as the intact protein, whereas on the modified liposomes plasminogen was present in degraded form. Also, the liposomes modified with PNIPAM in its extended conformation (below the lower critical solution temperature) appeared to adsorb less protein than those containing the 'collapsed' form of PNIPAM (above the LCST).

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *plasma --blood and lymphatics; *protein --analysis; *N-substituted polyacrylamides; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --fluorescence detection; *fluorescence spectroscopy --spectroscopic techniques: CB; *immunoblotting --analytical method; *immunoblotting --Detection/Labeling Techniques; *microscopy --analytical method; *microscopy --microscopy: CB; *SPEX Fluorolog 212 spectrometer --equipment

1999

Measurement of the absolute temporal coupling between DNA binding and base flipping.

Reich, Norbert O.; Allan, Barrett W.; Beechem, Joseph M.

Biochemistry, VOL. 38, NO. 17, April 27, 1999, PP. 5308-5314

The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 X 107 M-1 s-1 ltoreq kon ltoreq 2.25 X 107 M-1 s-1. Base-flipping kinetics (monitored using 2-aminopurine fluorescence intensity) were essentially synchronous with the binding kinetics, with less than a 4 ms delay between enzyme binding and target base flipping. The 4 ms delay translates into a base-flipping rate of at least 195 s-1, when the data are analyzed in terms of a sequential DNA binding and base-flipping reaction mechanism. Synchrony of binding and base flipping was only observed during the first 80% of the reaction, and an additional 20% base-flipping signal occurred well after DNA binding was complete. This additional 2AP fluorescence change, with an effective rate of 0.55 s-1, is an intramolecular isomerization reaction which greatly accelerates the dissociation of the enzyme from DNA. The correlation between the dissociation of the enzyme-DNA complex and the restacking of the extrahelical base also revealed a very tight coupling of these two events. Both dissociation and base restacking were found to be biphasic. These data are consistent with the following mechanism. The initial binding rate and base-flipping rates map very closely with previously determined pre-steady-state burst-rate kinetics for methyl transfer. Hence, binding, flipping, and methylation appear to occur in nearly a single concerted step. The bound complex then slowly isomerizes (0.1 s-1) to a distinct configuration that accelerates the product-release phase of the reaction. The product-release enzyme configuration dissociates from DNA approximately 8 times faster than the initial bound complex (0.18 s-1 vs 0.024 s-1). When the enzyme dissociates from the DNA along the product-release pathway, the target base remains in an extrahelical conformation and restacks at a rate of only 0.6 s-1. This "multicolor" fluorescence kinetic approach directly measures the absolute temporal correlation between DNA binding and base flipping, with millisecond timing resolution. The data reveal that even when the B-DNA structure is altered in a radical manner (e.g., via base flipping), enzymes can perform this operation in a highly efficient, if not completely concerted manner.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *oligonucleotides --purification; *oligonucleotides --synthesis; *DNA --analysis; *EcoRI DNA methyltransferase; *fluorescence spectrometry --analytical method; *fluorescence spectrometry --fluorophotometry: CB; *oligonucleotide synthesis --nucleic acid synthesis; *oligonucleotide synthesis --synthetic method; *Cyclone 8400 Plus DNA synthesizer --laboratory equipment; *Cyclone 8400 Plus DNA synthesizer --Milligen/Bioresearch; *Dynamax C18 reversed-phase PureDNA column --laboratory equipment; *Dynamax C18 reversed-phase PureDNA column --Rainin Instrument Co.; *HPLC high performance liquid chromatography --liquid chromatography; *HPLC high performance liquid chromatography --purification method; *SPEX 1681 Fluorolog spectrophotometer --laboratory equipment; *absolute temporal coupling --measurement; *base flipping; *enzyme-DNA interactions; *DNA binding

1999

Mapping the functional domains of BRCA1: Interaction of the ring finger domains of BRCA1 and BARD1.

Meza, J. E.; Brzovic, P. S.; King, M-C.; Klevit, R. E.

Journal of Biological Chemistry, VOL. 274, NO. 9, 1999, PP. 5659-5665

Breast cancer 1 (BRCA1) and BRCA1-associated RING domain 1 (BARD1) are multidomain proteins that interact in vivo via their N-terminal RING finger motif regions. To characterize functional aspects of the BRCA1/ BARD1 interaction, we have defined the structural domains required for the interaction, as well as their oligomerization state, relative stability, and possible nucleic acid binding activity. We have found that the RING finger motifs do not themselves constitute stable structural domains but are instead part of larger domains comprising residues 1-109 of BRCA1 and residues 26119 of BARD1. These domains exist as homodimers and preferentially form a stable heterodimer. Shorter BRCA1 RING finger constructs do not interact with BARD1 or with longer BRCA1 constructs, indicating that the heterodimeric and homodimer interactions are mediated by regions outside the canonical RING finger motif. Nucleic acid binding is a generally proposed function of RING finger domains. We show that neither the homodimers nor the heterodimer displays affinity for nucleic acids, indicating that the proposed roles of BRCA1 and BARD1 in DNA repair and/or transcriptional activation must be mediated either by other regions of the proteins or by additional cofactors.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BARD1; *BARD1 CONSTRUCT CLONING; *BARD1 N-TERMINAL CONSTRUCT EXPRESSION; *BARD1 N-TERMINAL CONSTRUCT PURIFICATION; *BINDING ASSAYS; *BRCA1; *BRCA1 CONSTRUCT CLONING; *BRCA1 FUNCTIONAL DOMAIN MAPPING; *BRCA1 N-TERMINAL CONSTRUCT EXPRESSION; *BRCA1 N-TERMINAL CONSTRUCT PURIFICATION; *BRCA1-ASSOCIATED RING DOMAIN 1; *BREAST CANCER 1 PROTEIN; *CHEMICAL MODIFICATION; *CHEMICAL MODIFICATION METHOD; *DNA REPAIR; *FLUORESCENCE SPECTROSCOPY; *FUNCTIONAL DOMAIN MAPPING; *GENE EXPRESSION-VECTOR TECHNIQUES; *GENETIC ENGINEERING METHOD; *ISOLATION-PURIFICATION TECHNIQUES: CB; *LABORATORY EQUIPMENT; *LIMITED PROTEOLYSIS; *MAPPING TECHNIQUES; *METHODOLOGY; *MOLECULAR GENETICS; *MULTIDOMAIN PROTEIN; *NUCLEIC ACID BINDING; *PURIFICATION METHOD; *RECOMBINANT DNA TECHNOLOGY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG 2 FLUOROMETER; *STOICHIOMETRY DETERMINATION; *TRANSCRIPTIONAL ACTIVATION

1999

Location and orientation of DODCI in lipid bilayer membranes: Effects of lipid chain length and unsaturation.

Periasamy, N.; Krishna, M.M.G.

Biochimica et Biophysica Acta, VOL. 1461, NO. 1, Nov. 9, 1999 , PP. 58-68

The location and orientation of a linear dye molecule, DODCI, in lipid bilayer membrane were determined by the effect of viscosity and refractive index of the aqueous medium on the fluorescence properties of the dye bound to the membrane. The membrane-bound dye is solubilized in two sites, one near the surface (short fluorescence lifetime) and another in the interior of the membrane (long lifetime). The ratio of the dye in the two locations and the orientation of the dye (parallel or perpendicular to the membrane) are sensitive to the lipid chain length and unsaturation in the alkyl chain. The fraction of the dye in the interior region is higher for short alkyl chains (C12 > C14 > 16 mchgt C18 apprxeq C20) and in unsaturated lipids (C14:1 > C14:0, C16:1 > C16:0). These experimental results are consistent with the general principle that the penetration of an amphiphilic organic molecule into the interior region of the membrane is more when the structure of the bilayer is more fluid-like.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *lipid bilayer membranes; *DODCI 3,3'-diethyloxadicarbocyanine iodide --analysis; *DODCI 3,3'-diethyloxadicarbocyanine iodide --Exciton; *spectrofluorophotometry --analytical method; *spectrofluorophotometry --photometry: CB; *SPEX Fluorolog 1681 T format spectrofluorophotometer --equipmen

1999

Large scale purification of detergent-soluble P-glycoprotein from Pichia pastoris cells and characterization of nucleotide binding properties of wild-type, Walker A, and Walker B mutant proteins

Senior, Alan E. .; Gimi, Khursheed; Gros, Philippe; Lerner-Marmarosh, Nicole; Urbatsch, Ina L

Journal of Biological Chemistry, VOL. 274, NO. 49, Dec. 3,. 1999, PP. 34711-34718

P-glycoprotein (Pgp; mouse MDR3) was expressed in Pichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wild-type mouse MDR3 Pgp were studied using 2'-(3')-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate. Both analogs were found to bind with Kd in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques.; *Pichia pastoris (Ascomycetes).; *Fungi; *Microorganisms; *Nonvascular Plants; *Plants.; *wild-type protein.; *ATP; *ATPase; *P-glycoprotein --detergent soluble protein; *P-glycoprotein --nucleotide binding properties; *P-glycoprotein --purification; *Walker A protein; *Walker B protein; *column chromatography --ion exchange chromatography; *column chromatography --purification method; *fluorometry --analytical method; *fluorometry --photometry: CB; *protein purification --purification method.; *protein purification --Isolation/Purification Techniques: CB; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --analytical method; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --electrophoretic techniques; *SPEX FLuorolog 2 fluorometer --equipment; *protein mutation.

1999

Interaction of 7-hydroxy-8-(phenylazo)1,3-naphthalenedisulfonate with bovine plasma albumin. Spectroscopic studies.

Srivastava, Sudha; Patel, Anant B.; Phadke, Ratna S.

Journal of Biological Chemistry, VOL. 274, NO. 31, July 30, 1999, PP. 21755-21762

Interaction of Orange G (OG) with bovine plasma albumin (BPA) has been investigated using NMR, UV-visible absorption, CD, and fluorescence techniques. The bound conformation of OG is a compact structure with N9-N10 bond in a non-planar syn conformation. The binding causes a decrease in the 478-nm absorption band of OG. The analysis of the binding isotherm generated from UV-visible absorption measurements gives a dissociation constant of 10 muM and stoichiometry 1:1 for BPAcntdotOG complex. Dissociation constant is invariant in the pH range 5.0-8.0 and is apprx20 times higher at pH 4.0 than its value at pH 7.0. Near and far UV-CD studies indicate alterations in the helical content and in the tertiary structure of the protein on complexation. The binding induces (-) and (+) CD at 335 nm and 465 nm, respectively. The binding also results into an increase in the steady state fluorescence anisotropy of OG without affecting emission maximum and quantum yield. Fluorescence data indicate that quenching of Trp fluorescence by OG is static in nature and OG selectively binds near Trp-135. Observation of similar rotational correlation time for BPA and BPAcntdotOG complex indicates that the overall globular structure of BPA remains unaltered on binding despite certain internal rearrangement in the protein structure.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *bovine plasma albumin --Armour Pharmaceutical Co.; *7-hydroxy-8-(phenylazo)1,3-naphthalenedisulfonate Orange G --Sigma; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *fluorescence spectrometry --analytical method; *fluorescence spectrometry --fluorophotometry: CB; *Jasco J-600 spectropolarimeter --equipment; *Jasco J-600 spectropolarimeter --Jasco; *NMR --analytical method; *NMR --imaging techniques; *NMR --spectroscopic techniques: CB; *Shimadzu spectrometer --equipment; *Shimadzu spectrometer --Shimadzu; *Spex Fluorolog 1681 T format spectrofluorophometer --equipment; *Spex Fluorolog 1681 T format spectrofluorophometer --Spex Fluorolog ; *UV-visible absorption --analytical method; *UV-visible absorption --spectroscopic techniques: CB; *Varian Unity Plus 600 MHz spectrometer --equipment; *Varian Unity Plus 600 MHz spectrometer --Varian; *7-hydroxy-8-(phenylazo)1,3-naphthalenedisulfonate-bovine plasma albumin interaction

1999

Identifying the site of initial tertiary structure disruption during apomyoglobin unfolding.

Loh, Stewart N.; Feng, Zhaoyang; Ha, Jeung-Hoi

Biochemistry, VOL. 38, NO. 44, Nov. 2, 1999, PP. 14433-14439

Structural characterization of protein unfolding intermediates (Kiefhaber et al. (1995) Nature 375, 513; Hoeltzli et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318), which until recently were thought to be nonexistent, is beginning to give information on the mechanism of unfolding. To test for apomyoglobin unfolding intermediates, we monitored kinetics of urea-induced denaturation by stop-flow tryptophan fluorescence and quench-flow amide hydrogen exchange. Both measurements yield a single, measurable kinetic phase of identical rate, indicating that the reaction is highly cooperative. A burst phase in fluorescence, however, suggests that an intermediate is rapidly formed. To structurally characterize it, we carried out stop-flow thiol-disulfide exchange studies of 10 single cysteine-containing mutants. Cysteine probes buried at major sites of helix-helix pairing revealed that side chains throughout the protein unpack and become accessible to the labeling reagent (5,5'-dithiobis (2-nitrobenzoic acid)) with one of two rates. Probes located at all helical-packing interfaces-except for one-become exposed at the rate of global unfolding as determined by fluorescence and hydrogen exchange measurements. In contrast, probes located at the A-E helical interface undergo complete thiol-disulfide exchange within the mixing dead time of 6 ms. These results point to the existence of a burst-phase unfolding intermediate that contains globally intact hydrogen bonds but locally disrupted side-chain packing interactions. Dissolution of secondary and tertiary structure are therefore not tightly coupled processes. We suggest that disruption of tertiary structure may be a stepwise process that begins at the weakest point of the native fold, as determined by native-state hydrogen-exchange parameters.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *apomyoglobin --analysis; *apomyoglobin --purification; *5,5'-dithiobis(2-nitrobenzoic acid) --labeling agent; *apomyoglobin purification --purification method; *apomyoglobin purification --Isolation/Purification Techniques: CB; *spectrofluorimetry --analytical method; *spectrofluorimetry --fluorometry: CB; *SPEX Fluorolog 3-21 spectrofluorimeter --equipment

1999

Fluorimetric nitrite analysis using 2,3-diaminonaphthalene: An improvement of the method.

Andre, J.-C.; Carre, M.-C.; Mahieuxe, B.; Viriot, M.-L.

Analusis, VOL. 27, NO. 10, Dec., 1999, PP. 835-838

One of the fluorimetric methods for nitrite determination is based on the reaction of nitrite ions with 2,3-diaminonaphthalene (DAN) to form fluorescent 1-(H)-naphthotriazole. The procedure is sensitive, simple and rapid. However, the DAN reagent is carcinogenic and its use in a large excess for the reaction might be a drawback for an extensive application. In particular to know if a stoechiometric quantity of the reagent might be sufficient to run the reaction, the method was re-examined. In fact, using the latter condition, it was still possible to get nitrite determination in the range 0 to 50 mug NO2-.L-1, with a rather smaller analysis time.

DESCRIPTOR(S)- *Chemistry; *Methods and Techniques; *nitrite --analysis; *2,3-diaminonaphthalene --reagent; *2,3-diaminonaphthalene --Aldrich; *spectrofluorimetry --analytical method; *spectrofluorimetry --fluorometry: CB; *SPEX 1681 Fluorolog spectrofluorimeter --equipment

1999

Fluorescence energy transfer as a probe for tetraplex formation: The i-motif.

Mergny, J-L.

Biochemistry, VOL. 38, NO. 5, 1999, PP. 1573-1581

The secondary structure of cytosine-rich oligodeoxynucleotides has been investigated with fluorescent probes. Intramolecular folding of an oligonucleotide into an i-DNA motif led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the DNA, respectively, provided that a suitable linker was chosen. The conjugation of the dyes to the oligonucleotide had an influence on the thermodynamics of i-motif formation as well as on the kinetics of folding. Intramolecular folding was demonstrated from the concentration independence of FRET over a wide concentration range. Folding of the oligonucleotide was confirmed by UV absorption melting experiments. The folding of the i-motif could be followed at concentrations as low as 50 pM. Fluorescence energy transfer can thus be used to reveal the formation of multistranded DNA structures.

DESCRIPTOR(S)- *ACCEPTOR; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CYSTEINE-RICH OLIGODEOXYNUCLEOTIDES; *DONOR; *DYE; *EUROGENTEC; *FLUORESCEIN; *FLUORESCENCE ENERGY TRANSFER; *FLUORESCENT PROBES; *HYSTERESIS ANALYSIS; *I-DNA MOTIF; *KONTRON; *KONTRON UVIKON 940 SPECTROPHOTOMETER; *LABORATORY EQUIPMENT; *MOLECULAR PROBE TECHNIQUES; *OLIGONUCLEOTIDE INTRAMOLECULAR FOLDING; *SECONDARY STRUCTURE ANALYSIS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG DM1B INSTRUMENT; *TETRAMETHYLRHODAMINE; *TETRAPLEX FORMATION; *TETRAPLEX FORMATION PROBE; *UV ABSORPTION MELTING EXPERIMENT

1999

Fluorescence binding assay for a small peptide based on a GFP fusion protein.

Daunert, Sylvia; Feliciano, Jessika; Lewis, Jennifer C.

Analytica Chimica Acta, VOL. 397, NO. 1-3, Oct. 4, 1999, PP. 279-286

A fluorescence binding assay was developed for a small peptide based on a fusion protein between the peptide and the green fluorescent protein, GFP. The assay employs genetic engineering methods to prepare the analyte-label (peptide-GFP) conjugate as a fusion protein in order to produce a one-to-one, homogenous population of labeled-peptide. Specifically, a plasmid was constructed in which the C-terminus of a model octapeptide was fused to the N-terminus of GFP. Following expression of the octapeptide-GFP fusion protein in Escherichia coli, an immunoassay was developed based on sequential binding of the free octapeptide and labeled-octapeptide to an anti-octapeptide antibody immobilized on a solid surface. The naturally fluorescent protein acts as a label to provide sensitive detection for peptides. To our knowledge, this is the first time that GFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for a peptide analyte.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae) --strain-JM109; *Bacteria; *Eubacteria; *Microorganisms; *octapeptide-green fluorescent protein fusion protein --assay; *octapeptide-green fluorescent protein fusion protein --isolation; *octapeptide-green fluorescent protein fusion protein --purification; *column purification --purification method; *column purification --Isolation/Purification Techniques: CB; *fluorescence binding assay --analytical method; *fluorescence binding assay --fluorophotometry: CB; *immunoassay --analytical method; *immunoassay --Analysis/Characterization Techniques: CB; *polymerase chain reaction --genetic engineering method; *polymerase chain reaction --in-situ recombinant gene expression detection; *protein isolation --isolation method; *protein isolation --Isolation/Purification Techniques: CB; *Perkin Elmer GeneAmp PCR System 2400 --equipment; *PhastSystem electrophoresis --equipment; *SDS polyacrylamide gel electrophoresis --analytical method; *SDS polyacrylamide gel electrophoresis --polyacrylamide gel electrophoresis; *SPEX Fluorolog-2 spectrofluorometer --equipment

1999

Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin.

Thomas, David D.; Prochniewicz, Ewa

Biochemistry, VOL. 38, NO. 45, Nov. 9, 1999, PP. 14860-14867

We have used spectroscopic probes ErIA and IAEDANS attached to Cys374 to compare the structural dynamics of yeast actin filaments with that of muscle actin, to understand the structural basis of the less productive interaction of yeast actin with myosin. Time-resolved phosphorescence anisotropy (TPA) of ErIA and steady-state fluorescence of IAEDANS were measured. TPA indicated more rapid rotational motion and more restricted angular amplitude in yeast actin. The fluorescence spectrum was less intense and more red-shifted in yeast actin, suggesting more exposure of the probe to solvent. These results indicate that the two actins differ substantially in the conformational dynamics of the C-terminal region. Binding of myosin S1 induced significantly different spectroscopic changes in TPA and fluorescence of muscle and yeast actin. As a result, the spectroscopic differences between the two actins were decreased by the addition of S1. These results suggest that yeast actin is less effectiveat activating myosin because of larger changes required in the structure of actin upon strong myosin binding. These results provide insight into the relationship between actomyosin dynamics and function, and they provide a useful framework for structure-function analysis of mutant yeast actin.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Muscular System (Movement and Support); *rabbit (Leporidae); *yeast (Fungi); *Animals; *Chordates; *Fungi; *Lagomorphs; *Mammals; *Microorganisms; *Nonhuman Mammals; *Nonhuman Vertebrates; *Nonvascular Plants; *Plants; *Vertebrates; *skeletal muscle --muscular system; *actin --analysis; *actin --fluorescence intensity; *actin --structural dynamics; *actin --transient phosphorescence anisotropy; *myosin --analysis; *myosin --functional actin interactions; *enzymatic digestion --cell disruption techniques; *enzymatic digestion --chemical method; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *ATPase assay --activity assays; *ATPase assay --bioassay method; *SPEX Fluorolog fluorimeter --equipment

1999

Determination of trace levels of polychlorinated biphenyls on reversed phase octadecyl bonded silica membranes.

Arruda, A. F.; Campiglia, A. D.,

Analytica Chimica Acta, VOL. 386, NO. 3, 1999, PP. 271-280

The determination of biphenyl, polychlorinated biphenyls, and Aroclors adsorbed on solid-phase extraction membranes was performed by room-temperature phosphorimetry. Three types of membranes were tested as substrates for phosphorescence emission. Several experimental parameters related to phosphorescence on solid supports were evaluated. The analytical figures of merit demonstrate the capability of detecting polychlorinated biphenyls at the sub-ng/ml level. The potential interference of polycyclic aromatic hydrocarbons was investigated. The combination of time discrimination and synchronous excitation techniques allowed the determination of polychlorinated biphenyls in the presence of polycyclic aromatic hydrocarbons. Although additional studies are required to prove solid phase extraction - room temperature phosphorimetry as a reliable analytical tool, our results reveal several promising features for screening polychlorinated biphenyls in water samples.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *AROCLORS; *BIOCHEMISTRY AND BIOPHYSICS; *BIPHENYL; *EXTRACTION METHOD; * FLUOROLOG-3 SPECTROFLUORIMETER; *HAMAMATSU; *ISOLATION-PURIFICATION TECHNIQUES: CB; *JOBINY-VON-SPEX; *LABORATORY EQUIPMENT; *METHODOLOGY; *PHOSPHORESCENCE; *PHOTOMULTIPLIER TUBE; *POLYCHLORINATED BIPHENYLS; *POLYCYCLIC AROMATIC HYDROCARBONS; *REVERSED PHASE OCTADECYL BONDED SILICA MEMBRANES; *ROOM-TEMPERATURE PHOSPHORIMETRY; *SOLID PHASE EXTRACTION

1999

Defining the core structure of the alpha-lactalbumin molten globule state

Raleigh, Daniel P. .; Boice, Judith A.; Demarest, Stephen J.; Fairman, Robert

Journal of Molecular Biology, VOL. 294, NO. 1, Nov. 19,. 1999, PP. 213-221

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the noncooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Radiation Biology.; *human alpha-lactalbumin --core structure; *human alpha-lactalbumin --molten globule state.; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *deletion analysis --analytical method; *far UV circular dichroism --radiobiology method; *fluorescence experiments --analytical method; *fluorescence measurements --analytical method; *fluorescence measurements --spectroscopic techniques: cb; *near UV circular dichroism --radiobiology method; *peptide synthesis --synthesis/modification techniques; *peptide synthesis --synthetic method; *sedimentation equilibrium --centrifugation techniques; *sedimentation equilibrium --purification method; *urea denaturation experiments --analytical method.; *Aviv Model 62A circular dichroism spectrometer --laboratory equipment; *ANS binding experiments 1-anilinonaphthalene-8-sulfonate binding experiments --analytical method; *ISA Fluorolog spectrometer --laboratory equipment; *ISA Fluorolog spectrometer --ISA

1999

Charged microbeads are not transported across the human stratum corneum in vitro by short high-voltage pulses.

Chen, T.; Langer, R.; Weaver, J. C.

Chen, T.; Langer, R.; Weaver, J. C., Bioelectrochemistry and Bioenergetics, VOL. 48, NO. 1, 1999, PP. 181 - 192

 

1999

Characterization of the interactions between human cdc25C, cdks, cyclins and cdk-cyclin complexes.

Morris, M. C.; Divita, G.

Journal of Molecular Biology, VOL. 286, NO. 2, 1999, PP. 475-487

We have overexpressed and purified human dual-specificity phosphatase cdc25C from a prokaryotic expression system at high levels and in a soluble, active form, and have studied and quantified its potential to interact with cdks, cyclins and preformed cdk-cyclin complexes by fluorescence spectroscopy and size-exclusion chromatography. Our data indicate that human cdc25C forms stable complexes, through hydrophobic contacts, with cdk and cyclin monomers, as well as with preformed cdk-cyclin complexes. In vitro, cdc25C interacts with cyclin monomers with high affinity, with tenfold less affinity with cdks, and with intermediate affinity with cdk-cyclin complexes. Moreover, changes observed in the intrinsic fluorescence of cdks, cyclins and cdk-cyclin complexes upon interaction with cdc25C are indicative of concomitant conformational changes within cdks and cyclins. From our results, we propose that in vitro, in the presence of monomeric cdks and cyclins, cdc25C forms stable ternary complexes, first through a high affinity interaction with a cyclin, which may then help target cdc25C towards a cdk. We discuss the biological relevance of our results and propose that a similar, two-step mechanism of interaction between cdc25C and cdk-cyclin complexes may occur in vivo.

DESCRIPTOR(S)- *ACTIVITY ASSAYS; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CYCLIN DEPENDENT KINASE-CYCLIN COMPLEX; *CYCLIN DEPENDENT KINASES; *CYCLINS; *ESCHERICHIA COLI; *EXPRESSION SYSTEM; *FLUORESCENCE SPECTROSCOPY; *GEL FILTRATION HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; *GEL FILTRATION HPLC; *HUMAN CDC25C PROTEIN; *LABORATORY EQUIPMENT; *LIQUID CHROMATOGRAPHY; *METHODOLOGY; *PARA-NITROPHENYL PHOSPHATE ASSAY; *PHOSPHATASE; *PROTEIN-PROTEIN INTERACTIONS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX II JOBIN YVON FLUOROLOG SPECTROFLUOROMETER; *STRAIN-AR68

1999

Characterization and binding specificity of the monomeric STAT3-SH2 domain.

Haan, S.; Hemmann, U.; Hassiepen, U.; Schaper, F.; Schneider-Mergener, J.; Wollmer, A.; Heinrich, P. C.; Groetzinger, J.

Journal of Biological Chemistry, VOL. 274, NO. 3, 1999, PP. 1342-1348

Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction. STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains. The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus. Here we describe the expression, purification, and refolding of the STAT3-SH2 domain. Proper folding of the isolated protein was proven by circular dichroism and fluorescence spectroscopy. The STAT3-SH2 domain undergoes a conformational change upon dimerization. Using an enzyme-linked immunosorbent assay we demonstrate that the monomeric domain binds to specific phosphotyrosine peptides. The specificity of binding to phosphotyrosine peptides was assayed with the tyrosine motif encompassing Tyr-705 of STAT3 and with all tyrosine motifs present in the cytoplasmic tail of the signal transducer gp130.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *AVIV; *BINDING SPECIFICITY; *BIOCHEMISTRY AND BIOPHYSICS; *CHARACTERIZATION; *CIRCULAR DICHROISM; *DETECTION-LABELING TECHNIQUES; *ELISA; *ESCHERICHIA COLI; *EXPRESSION; *EXPRESSION SYSTEM; *FLUORESCENCE SPECTROSCOPY; *GLYCOPROTEIN 130; *GP130; *LABORATORY EQUIPMENT; *METHODOLOGY; *MONOMERIC SH2 DOMAIN; *MURINE STAT3 CDNA; *MURINE STAT3 COMPLEMENTARY DNA; *PEPTIDE BINDING ASSAY; *PURIFICATION; *REFOLDING; *SIGNAL TRANSDUCER; *SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG 211 PHOTON-COUNTING SPECTROFLUOROMETER; *SPEX INDUSTRIES; *STAT3; *STRAIN-BL21(DE3)PLYSS; *62DS CIRCULAR DICHROISM SPECTROMETER

1999

Binding of the transition state analog MgADP-fluoroaluminate to F-1-ATPase.

Nadanaciva, S.; Weber, J.; Senior, A. E.

Journal of Biological Chemistry, VOL. 274, NO. 11, 1999, PP. 7052 - 7058

Escherichia coli F-1-ATPase from mutant beta-Y331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F-1-ATPase.

DESCRIPTOR(S)- *AMICO-BOWMAN 2 SPECTROFLUOROMETER; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CATALYTIC SITE BINDING ANALYSIS; *ESCHERICHIA COLI; *F-1-ATPASE; *LABORATORY EQUIPMENT; *MAGNESIUM-ADP; *MAGNESIUM-ADP-FLUOROALUMINATE; *METHODOLOGY; *SPECTROFLUOROMETRY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG 2 SPECTROFLUOROMETER; *TRANSITION STAE ANALOG

1999

Autonomous folding of a C-terminal inhibitory fragment of Escherichia coli isoleucine-tRNA synthetase.

Miller, W. Todd; Michaels, John-Edward A.; Shiba, Kiyotaka

Biochimica et Biophysica Acta, VOL. 1433, NO. 1-2, Aug. 17, 1999, PP. 103-109

We previously reported that C-terminal fragments of Escherichia coli Ile-tRNA synthetase, a monomeric enzyme of 939 amino acids, act as dominant negative inhibitors of the wild-type enzyme in vivo and in vitro. Our experiments suggested that it is possible to block the functional assembly of a monomeric protein by interfering with the folding pathway. We postulated that the inhibitory C-terminal fragments fold autonomously, and in the presence of full-length Ile-tRNA synthetase, trap the N-terminal portion of polypeptide in an unproductive complex. Here, we report the results of experiments aimed at understanding the mechanism of dominant negative inhibition. We have carried out biophysical experiments on fragment 585-939 of Ile-tRNA synthetase, which we previously determined to be the minimal inhibitory unit. Circular dichroism and fluorescence spectroscopy indicate that this fragment forms a compact and stable structure in solution. The secondary structure of this fragment is predominantly alpha-helical, consistent with the crystal structure of Ile-tRNA synthetase from another organism. The C-terminal fragment is capable of forming native-like secondary and tertiary structure after refolding from guanidine HCl. Taken together, the results are consistent with the hypothesis that the inhibitory fragment of Ile-tRNA synthetase forms an independent folding unit.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *isoleucine-tRNA synthase isoleucine-transfer RNA synthase --autonomous folding; *isoleucine-tRNA synthase isoleucine-transfer RNA synthase --characterization; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: cb; *Aviv 62 A-DS circular dichroism spectropolarimeter --laboratory equipment; *Spex Fluorolog spectrofluorimeter --laboratory equipment; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --gel electrophoresis; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --purification method

1999

A dual-probe fluorescence method to examine selective perturbations of membrane permeability by melittin.

Lafleur, Michel; El Jastimi, Rachida

Biospectroscopy, VOL. 5, NO. 3, 1999, PP. 133-140.

A new fluorescence method has been developed to measure simultaneously and independently the release of fluorophores from two vesicle populations. Calcein and sulforhodamine B were used as a probe couple: the leakage of these probes from vesicles can be recorded independently since they can be excited simultaneously at 510 nm, and their individual fluorescence can be isolated by measuring the fluorescence signal at 525 and 590 nm, using a T-shape fluorometer. Controls show that both probes are suitable for the leakage assay based on fluorescence self-quenching, that they do not interact physically or chemically at the concentrations used in the method, and that they leak in a similar fashion from a given vesicle type. This dual-probe technique is applied to examine the specificity of the release relative to the cholesterol content of the vesicles for melittin, a toxin. This new approach shows in a straightforward manner that melittin-induced release for a given population can be modulated by the presence of vesicles with another lipid composition and this competitive release is associated with a preferential distribution of the peptide on the targeted vesicles.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Toxicology; *calcein --reagent; *calcein --Molecular Probes; *cholesterol; *melittin --analysis; *melittin --membrane permeability; *melittin --toxin; *sulforhodamine B --reagent; *sulforhodamine B --Molecular Probes; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CB; *SPEX Fluorolog-2 spectrometer --equipment