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

タイトル

著者

掲載誌

概要

1998

Tryptophan substitutions surrounding the nucleotide in catalytic sites of F-1-ATPase.

Weber, J.; Wilke-Mounts, S.; Hammond, S. T.; Senior, A. E.

Biochemistry, VOL. 37, NO. 35, 1998, PP. 12042-12050

Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F-1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp- 153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, V-max MgATP hydrolysis.

DESCRIPTOR(S)- *AMINCO; *AMINCO BOWMAN 2 SPECTROFLUOROMETER; *ANALYTICAL METHOD; *CATALYTIC SITES; *ENZYMOLOGY; *EQUIPMENT; *ESCHERICHIA-COLI F-1-ATPASE; *FLUORESCENCE SPECTROSCOPY; *GEL ELECTROPHORESIS; *GENETIC METHOD; *METHODOLOGY; *MUTAGENESIS; *NUCLEOTIDE; *PROTEIN ENGINEERING; *SDS-GEL ELECTROPHORESIS; *SEPARATION METHOD; *SITE-DIRECTED MUTAGENESIS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX; *SPEX FLUOROLOG 2 SPECTROFLUOROMETER; *TRYPTOPHAN SUBSTITUTIONS

1998

Thermal Degradation of Hair

Jachowicz, J. and McMullen, R. - International Specialty Products, Wayne, N.J.

Part. I Effect of Curling Irons Part. ‡U. Effect of Selected Polymers and Surfactants J. Soc. Cosmet. Chem. July/August (1998). (Accepted for Publication)

 

1998

The location of fluorescence probes with charged groups in model membranes.

Kachel, K.; Asuncion-Punzalan, E.; London, E.

Biochimica et Biophysica Acta, VOL. 1374, NO. 1-2, 1998, PP. 63-76

The location of commonly used charged fluorescent membrane probes in membranes was determined in order to: (1) investigate the relationship between the structure of hydrophobic molecules and their depth within membranes; and (2) aid interpretation of experiments in which these fluorescent probes are used to examine membrane structure. Membrane depth was calculated using parallax analysis, a method in which the quenching induced by lipids carrying a nitroxide group at different locations in the membrane is compared. Shallow locations were found for xanthene dyes (fluorescein, eosin, Texas Red and rhodamine) both in free form and when attached either to the headgroup of phospholipids or long hydrocarbon chains. The exact structure of the xanthene and the nature of its linkage to lipid had only a modest effect on membrane location, which ranged between 19 and 24 ANG from the center of the bilayer in a charged state. Thus, the location of these fluorophores largely reflects their intrinsic properties rather than the nature of the groups to which they are attached. Furthermore, cationic and anionic xanthene derivatives had similar depths, indicating the type of charge does not have a large effect on depth. Consistent with this conclusion, shallow locations were also found for other hydrocarbon chain-linked cationic (acridine orange and styrylpyridinium) and anionic (coumarin, anilinonaphthalenesulfonic acid (ANS), and toluidinylnaphthalenesulfonic acid (TNS)) charged probes. These all located at 16-18 ANG from the bilayer center. We conclude that both anionic and cationic molecules that are otherwise hydrophobic predominantly occupy shallow locations within the polar headgroup region of the bilayer no matter how hydrophobic the molecule to which they are linked. This depth is significantly shallower than that occupied by most previously studied uncharged polar molecules that locate near the membrane surface. Consistent with this conclusion, a 2-4 ANG deeper location was found for xanthene probes with no net charge. In other experiments, methods to avoid chemical reactions that can distort the measurement of depth by fluorescence quenching were developed.

DESCRIPTOR(S)- *ANALYSIS; *ANALYTICAL METHOD; *AVANTI POLAR LIPIDS; *BIOCHEMISTRY AND BIOPHYSICS; *FLUORESCEIN; *LABORATORY EQUIPMENT; *LIQUID CHROMATOGRAPHY; *MEMBRANE DEPTH; *MEMBRANES; *METHODOLOGY; *MODEL MEMBRANES; *N-(LISSAMINE RHODAMINE B SULFONYL)-1,2-DEHEXADECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE; *N-(TEXAS RED SULFONYL)-1,2-DEHEXADECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE; *N-(5-FLUORESCEINT- HIOCARBAMOYL)-1,2-DEHEXADECANOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE; *OCTADECYL ACRIDINE ORANGE; *SPEX 212 FLUOROLOG SPECTROFLUORIMETER; *THIN LAYER CHROMATOGRAPHY; *XANTHENE DYES; *1-ANILINONAPHTHALENE-8-SULFONIC ACID; *1-PALMITOYL-2-(12-DOXYL)STEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE; *1-PALMITOYL-2-(5-DOXYL)STEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE; *1-PYRENESULFONIC ACID; *1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE; *2-(P-TOLUIDINYL)NAPHTHALENE-6-SULFONIC ACID; *3-HEXADECANOYL-7-HYDROXYCOUMARIN; *5-(N-DODECANOYL)AMINOFLUORESCEIN; *5-(N-HEXADECANOYL)AMINOEOSIN; *5-(N-HEXADECANOYL)AMINOFLUORESCEIN

1998

Structural changes in human tear lipocalins associated with lipid binding.

Gasymov, O. K.; Abduragimov, A. R.; Yusifov, T. N.; Glasgow, B. J.

Biochimica et Biophysica Acta, VOL. 1386, NO. 1, 1998, PP. 145-156

Structural and conformational changes in tear lipocalins were detected in association with ligand binding and release. Circular dichroism measurements demonstrated that ligand binding induces beta structure formation, aromatic side chain asymmetry, and a more rigid state in tear lipocalins (TL). The exposure of the tyrosyl component is less in apo-TL than in holo-TL. The sole tryptophan residue, Trp-17, is buried in both holo- and apo-TL. The steady state exposure of Trp-17 is the same in holo- and apo-TL, but the dynamic exposure is two-fold greater in apo-TL. Maneuvers to unfold the protein with urea or incubation in an acidic environment resulted in increased exposure of aromatic amino acids. Electron paramagnetic resonance studies verified that lipids are liberated from TL in an acidic environment. Acidic pH promotes conformational changes in TL involving aromatic residues, particularly the conserved residue Trp-17. These changes are associated with lipid release. The liberation of lipid from the cavity of TL under acidic conditions involves a molten globule state of the protein. We postulate that TL, exposed to the steep surface pH gradient that exists at lipid-aqueous interfaces, would release lipid in association with a molten globule transition. The data suggest a plausible regulatory mechanism for lipid delivery from lipocalins at the tear film surface.

DESCRIPTOR(S)- *ANALYSIS; *ANALYTICAL METHOD; *AROMATIC AMINO ACIDS; *BIOCHEMISTRY AND BIOPHYSICS; *CIRCULAR DICHROISM; *COLUMN CHROMATOGRAPHY; *DMSO; *EPR SPECTROSCOPY; *FLUORESCENCE SPECTROSCOPY; *GEL ELECTROPHORESIS; *HUMAN TEAR LIPOCALINS; *ION EXCHANGE COLUMN CHROMATOGRAPHY; *JASCO; *JASCO 600 SPECTROPOLARIMETER; *JOBIN YVON; *JOBIN YVON-SPEX FLUOROLOG-3 SPECTROFLUOROMETER; *KONTRON; *KONTRON 820 SPECTROPHOTOMETER; *LABORATORY EQUIPMENT; *LIGAND BINDING; *LIPID BINDING; *LIPID-AQUEOUS INTERFACE; *LIQUID CHROMATOGRAPHY; *METHODOLOGY; *MOLTEN GLOBULE STATE; *PERKIN-ELMER; *PERKIN-ELMER LS-5; *PH; *PHOTOMETRY: CB; *PURIFICATION; *PURIFICATION METHOD; *SDS-PAGE; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SIGMA; *SIZE EXCLUSION CHROMATOGRAPHY; *SPECTROSCOPIC TECHNIQUES: CB; *SPECTROSCOPY; *STRUCTURE; *VARIAN; *VARIAN E-109 SPECTROMETER; *8-ANILINO-1-NAPHTHALENESULFONATE

1998

Stopped-flow fluorescence study of precatalytic primer strand base-unstacking transitions in the exonuclease cleft of bacteriophage T4 DNA polymerase.

Otto, M. R.; Bloom, L. B.; Goodman, M. F.; Beechem, J. M.

Biochemistry, VOL. 37, NO. 28, 1998, PP. 10156-10163

DNA polymerases are complex enzymes which bind primer-template DNA and subsequently either extend or excise the terminal nucleotide on the primer strand. In this study, a stopped-flow fluorescence anisotropy binding assay is combined with real-time measurements of a fluorescent adenine analogue (2-aminopurine) located at the 3'-primer terminus. Using this combined approach, the exact time course associated with protein binding, primer terminus unstacking, and base excision by the 3' fwdarw 5' exonuclease of bacteriophage T4 (T4 pol) was examined. T4 pol binding and dissociation kinetics were found to obey simple kinetics, with identical on rates (k-on = 4.6 times 10-8 m-1 s-1) and off rates (k-off = 9.3 s-1) for both single-stranded primers and double-stranded primer-templates (at 100 mu-M Mg-2+). Although the time course for T4 pol-DNA association and dissociation obeyed simple kinetics, at suboptimal Mg-2+ concentrations (e.g., 100 mu-M), non-first-order sigmoidal kinetics were observed for the base-unstacking reaction of the primer terminus in double-stranded primer-templates. The observed sigmoidal kinetics for base unstacking demonstrate that T4 pol is a hysteretic enzyme (Frieden, C. (1970) J. Biol. Chem. 245, 5788-5799) and must exist in two DNA bound conformations which differ greatly in base-unstacking properties. A Mg-2+-dependent time lag of 10 ms is observed between primer-template binding and the beginning of the unstacking transition, which is 50% complete at 22 +- 1 ms after addition of 100 mu-M Mg-2+. Following the hysteretic lag, a simple first-order primer terminus unstacking rate of 130 s-1 is resolved, which is protein and Mg-2+ concentration-independent. For the processing of single-stranded primers, all kinetic complexity is lost, and T4 pol binding and primer end base-unstacking kinetics can be superimposed. These data reveal that the kinetic processing of double-stranded primer-template DNA by T4 pol is much more complex than that of single-stranded primers, and suggest that the intrinsic "switching rate" between the polymerase and exonuclease sites may be much faster than previously proposed.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BACTERIOPHAGE T4 DNA POLYMERASE; *DOUBLE-STRANDED PRIMER-TEMPLATE DNA; *ENZYMOLOGY; *EXONUCLEASE CLEFT; *KINETICS; *LABORATORY EQUIPMENT; *METHODOLOGY; *PRECATALYTIC PRIMER STRAND BASE-UNSTACKING TRANSITIONS; *SPEX; *SPEX 1681 FLUOROLOG SPECTROFLUORIMETER; *STOPPED-FLOW FLUORESCENCE ANISOTROPY BINDING ASSAY

1998

Spectroscopy and non-radiative processes in Gd-3+, Eu-3+ and Tb-3 tropolonates.

Santos, B. S.; Donega, C. D. M.; Sa, G. C. D.; Oliveira, L. F. C. D.; Santos, P. S.

Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy, VOL. 54, NO. 13, 1998, PP. 2237-2245

Complexes of Eu-3+, Gd-3+, and Tb-3+ with tropolone (tropolone = 2-hydroxy-2,4,6-cycloheptatrien-1-one) were synthesized and characterized by C and H elemental analysis, infrared vibrational spectroscopy, X-ray powder diffraction analysis and Raman vibrational spectroscopy. The results show that the complexes are isostructural and have the formula (Ln(trop)-3), where Ln = Eu-3+, Gd-3+ and Tb-3+ and trop=tropolone. The characteristic luminescence of the Eu-3+ and Tb-3+ ions was not observed either upon ligand excitation (300-380nm) or upon direct f-f excitation, not even at 4.2 K. In order to understand this phenomenon the spectroscopic properties of the Gd(trop), complex (absorption spectra at 298 K, excitation and emission spectra from 4.2 to 298 K and decay time measurements from 20 to 298 K) were investigated. The emission spectra show three distinct bands, which are ascribed to ligand electronic states: a singlet state, S-1 (pi-pi-*), at 24270 cm-1, and two triplet states, T-1 and T-2, at 15630cm-1 and 16610cm-1, respectively. Both triplet states are situated below the emitting states of the Eu-3+ and Tb-3+ ions (5D-o and 5D-4, respectively), thus suppressing the luminescence from these states by back-transfer.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BRUKER; *BRUKER IF566 FTIR SPECTROPHOTOMETER; *CHEMISTRY; *ELEMENTAL ANALYSIS; *EUROPIUM TROPOLONATE; *GADOLINIUM TROPOLONATE; *IR VIBRATIONAL SPECTROSCOPY; *JOBIN-YVON; *JOBIN-YVON U-1000 RAMAN SPECTROMETER; *LABORATORY EQUIPMENT; *METHODOLOGY; *NON-RADIATIVE PROCESSES; *PERKIN-ELMER; *PERKIN-ELMER LAMBDA 6 SPECTROPHOTOMETER; *QUALITATIVE-QUANTITATIVE TECHNIQUES; *RAMAN VIBRATIONAL SPECTROSCOPY; *SHIMADZU; *SHIMADZU TGA-50 GRAVIMETRIC APPARATUS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG ; *SPEX FLUOROLOG DM3000F SPECTROFLUOROMETER; *TERBIUM TROPOLONATE; *THERMOGRAVIMETRY; *UV-VISIBLE ELECTRONIC ABSORPTION SPECTROSCOPY; *X-RAY ANALYSIS; *X-RAY POWDER DIFFRACTION ANALYSI

1998

Solid-matrix fluorescence and phosphorescence detection and characterization of benzo(a)pyrene-DNA adducts with Whatman No. 1 and Whatman 1PS filter paper.

Tjioe, S. W.; Hurtubise, R. J.

VOL. 52, NO. 3, 1998, PP. 414-419

Benzo(a)pyrene (B(a)P) is a potential carcinogen and mutagen, and it has been studied very extensively. In humans, B(a)P can be metabolized and becomes chemically bonded to DNA. A variety of methods are needed for the characterization of B(a)P-DNA adducts because of the structural complexity of the adducts. In this work, several solid-matrix luminescence methods were developed for the characterization of the B(a)P-DNA adducts. Both solid-matrix fluorescence and solid-matrix phosphorescence could be obtained from the adducts on Whatman No. 1 and Whatman 1PS filter paper at nanogram levels. Thallium nitrate proved to be an effective heavy atom in enhancing the solid-matrix phosphorescence of the adducts. By selective excitation of the phosphorescence of the adducts, it was possible to excite different forms of the adducts. With moisture quenching of the solid-matrix phosphorescence of the B(a)P-DNA adducts, it was feasible to selectively detect quasi-intercalated adducts. With Whatman 1PS filter paper, a unique method was developed for the solid-matrix fluorescence detection of adducts externally bound to DNA by the hydrophobic interaction of the adducts with the 1PS paper.

DESCRIPTOR(S)- *ANALYSIS; *ANALYTICAL METHOD; *BENZO(A)PYRENE; *BENZO(A)PYRENE-DNA ADDUCTS; *CARCINOGEN; *CHARACTERIZATION; *DETECTION; *DNA; *EQUIPMENT; * FLUOROLOG SPECTROFLUOROMETER; *METHODOLOGY; *MOLECULAR GENETICS; *MUTAGEN; *PERKIN-ELMER; *PERKIN-ELMER LS 5 LUMINESCENCE SPECTROMETER; *PHOSPHORESCENCE ENHANCER; *PHOSPHORESCENCE EXCITATION-EMISSION SPECTRA; *PHOTOMETRY; *SOLID-MATRIX FLUORESCENCE SPECTROMETRY; *SPECTROSCOPIC TECHNIQUES; *SPEX INDUSTRIES; *THALLIUM NITRATE; *WHATMAN; *WHATMAN IPS FILTER PAPER; *WHATMAN NUMBER 1 FILTER PAPER

1998

Set of secondary emission standards for calibration of the spectral responsivity in emission spectroscopy.

Gardecki, J. A.; Maroncelli, M.

Applied Spectroscopy, VOL. 52, NO. 9, 1998-09, PP. 1179-1189

A set of six secondary emission standards based on the commercially-available fluorophores tryptophan, small alpha -NPO, tetraphenylbutadiene, coumarin 153, DCM and LDS 751, is proposed for the purpose of checking and calibrating the spectral response of fluorescence spectrometers. The set of standards covers the range 300-800 nm with significant spectral overlap between fluorophores. Standard emission spectra were acquired by averaging the data obtained from three independently calibrated fluorescence spectrometers ( viz. , a Spex Industries Fluorolog F212, a Photon Technologies Inc. QuantaMaster QM-1 and a Spex Industries Fluorolog 3) and a method for generating a wavelength-dependent correction file was developed. Spectra were acquired on solutions of the fluorophores in H 2 O (tryptophan), cyclohexane (tetraphenylbutadiene) or methanol ( small alpha -NPO, coumarin 153, DCM and LDS 751) in each instance. Technical spectra of the fluorophores agreed to plus or minus 6% average absolute deviation and it was concluded that the differences between spectra acquired on the different spectrometers were primarily due to variations in the spectral distributions of the calibrated tungsten-halogen lamps and methods used in their individual correction. The spectrum of quinine sulfate based upon a correction file generated by use of the proposed method was in good agreement with NBS-reported spectrum.

1998

Separation of polycyclic aromatic hydrocarbon metabolites by gamma-cyclodextrin-modified micellar electrokinetic chromatography with laser-induced fluorescence detection.

Smith, C. J.; Grainger, J.; Patterson, D. G., Jr

Journal of Chromatography A, VOL. 803, NO. 1-2, 1998, PP. 241-247

Using a modified micellar buffer consisting of gamma-cyclodextrin (gamma-CD) and sodium dodecyl sulfate (SDS), we have obtained separations of hydroxy-polycyclic aromatic hydrocarbons (hydroxyPAHs). These compounds are oxidative products of mammalian PAH metabolism. The analytes were detected with a commercial laser-induced fluorescence (LIF) detector. A number of hydroxyPAH isomers could be separated by changes in gamma-CD concentration. Baseline resolution of 12 hydroxyPAHs was obtained using 30 mM borate, 60 mM SDS and 40 mM gamma-CD. The particular site substitution of the hydroxy group can produce changes in the hydroxyPAH fluorescence spectrum, and the effect of optical filter selection was studied for the LIF detection. The mass detection limits were in the (0.08-0.5) times 10-15 mol range. To our knowledge, this is the first report of the separation of metabolic products of PAHs (and several positional isomers) using gamma-CD and micellar electrokinetic chromatography.

DESCRIPTOR(S)- *ADVANCED SEPARATION TECHNOLOGIES; *ALPHA-NAPHTHOL; *BECKMAN; *BETA-NAPHTHOL; *BORATE; *BUFFER; *CHROMATOGRAPHIC TECHNIQUES; *ELECTROKINETIC CHROMATOGRAPHY LASER-INDUCED FLUORESCENCE DETECTION; *EQUIPMENT; *GAMMA-CYCLODEXTRIN; *ISA JOBIN YVON; *LASER-INDUCED FLUORESCENCE DETECTOR; *METHODOLOGY; *METHODSFINDER; *P-ACE 2200; *POLLUTANT; *POLLUTION; *POLYCYCLIC AROMATIC HYDROCARBON METABOLITES; *SDS; *SEPARATION; *SEPARATION METHOD; *SIGMA; *SPEX FLUOROLOG TAU-2 SPECTROFLUOROMETER; *1-HYDROXYBENZ(A)ANTHRACENE; *1-HYDROXYBENZO(A)PYRENE; *1-HYDROXYBENZO(B)FLUORANTHENE; *1-HYDROXYPYRENE; *2-HYDROXYINDENO(1,2,3-CD)PYRENE; *3-HYDROXYBENZ(A)ANTHRACENE; *3-HYDROXYBENZO(A)PYRENE; *3-HYDROXYCHRYSENE; *7-HYDROXYBENZO(A)PYRENE; *9-HYDROXYBENZO(A)PYRENE

1998

Probing DNA sequences in solution with a monomer-excimer fluorescence color change.

Paris, P. L.; Langenhan, J. M.; Kool, E. T.

Nucleic Acids Research, VOL. 26, NO. 16, 1998, PP. 3789-3793

The use of a simple fluorescent nucleoside analogue in detection of point mutations by hybridization in solution is described. Pyrene is placed at 3' and 5' ends of a pair of oligodeoxynucleotide probes via a phosphoramidite derivative of deoxyribose with this fluorophore attached at the 1' position, replacing a DNA base. Adjacent binding of dual probes containing this fluorophore to a complementary target sequence results in a pronounced spectral change from blue pyrene monomer emission (lambda-max = 381 398 nm) to greenwhite excimer emission (lambda-max = 490 nm). Optimization of the relative binding positions of the two probes shows that the greatest spectral change occurs when they bind with partial end overlap. In optimum orientation, the monomer emission band for the probes decreases intensity by as much as a factor of seven and the excimer band increases up to 40-fold on binding a complementary target. Application to the detection of a single-base point mutation in solution is described.

DESCRIPTOR(S)- *ANALYTICAL METHOD; *APPLIED BIOSYSTEMS; *APPLIED BIOSYSTEMS 392 SYNTHESIZER; *DNA; *FLUORESCENCE DETECTION; *FLUORESCENCE IN SITU HYBRIDIZATION; *FLUORESCENT NUCLEOSIDE ANALOGUE; *GEL ELECTROPHORESIS; *IN SITU HYBRIDIZATION; *LABORATORY EQUIPMENT; *METHODOLOGY; *MOLECULAR GENETICS; *MONOMER-EXCIMER FLUORESCENCE COLOR CHANGE; *NMR; *POINT MUTATIONS; *POLYACRYLAMIDE GEL ELECTROPHORESIS; *PROBING DNA SEQUENCES; *PROTEIN SYNTHESIS; *PURIFICATION METHOD; *PYRENE; *SPECTROSCOPIC TECHNIQUES: CB; * SPEX-FLUOROLOG-2 SERIES FLUOROMETER; *SYNTHESIS-MODIFICATION TECHNIQUES; *SYNTHETIC METHOD; *UV TRANSILLUMINATOR

1998

Pigment-pigment interactions in thylakoids and LHCII of chlorophyll a-c containing alga Pleurochloris meiringensis: Analysis of fluorescence-excitation and triplet-minus-singlet spectra.

Buechel, C.; Naqvi, K. R.; Melo, T. B.

Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy, NO. 5, 1998, PP. 719-726

Time-resolved triplet-minus-singlet (TmS) difference spectra, DELTA-A(lambda; t), fluorescence excitation spectra, X(lambda), and absorption spectra, A(lambda), are used for probing pigment-pigment interactions in the thylakoids (Chla/c-ThyI) and isolated light-harvesting complexes associated with photosystem II (Chla/c-LHCII) of the alga Pleurochloris meiringensis, whose chromophores comprise chlorophyll a (Chla), chlorophyll c (Chlc), and several carotenoids. The data provide information about interactions between Car*-and-Chla-0, Chla dag -and-Car-0, Car dag -and-Chla-0 (where the abbreviation Car stands for carotenoid, an asterisk and a dagger denote singlet and triplet excitation, respectively, and the superscript 0 denotes a molecule in the ground state). In Chla/c-ThyI, the efficiency of Car* fwdarw Chla* transfer (vphi-LH), determined by comparing A(lambda) and X(lambda), is slightly less than unity (ca. 0.85), whereas the efficiency of Chla dag fwdarw Car dag transfer of triplet energy (vphi-TT) must be much closer to unity, since no long-lived Chla dag could be detected; an interaction between Car dag and Chla-0, already familiar from investigations concerning the TmS spectra of the trimers and aggregates of Chla/b-LHCII (the light-harvesting complex associated with the photosystem II of higher plants), which manifests itself through a depletion signal (in the Q-y region of Chla) decaying at the same rate as the Car TmS signal, is observed, and explained likewise. In Chla/c-LHCII, both efficiencies are found to be much lower; the drastic reduction in the two yields is attributed to the perturbation of the native molecular architecture of the complex by the detergent used in the isolation procedure. The overall TmS signal from Chla/c-LHCII can be decomposed into two contributions, DELTA-A(lambda; t) = DELTA-1A(lambda; t)+DELTA-2A(lambda; t), where DELTA-1A(lambda; t) with a lifetime of about 8 mu-s; DELTA-2A(lambda; t), which persists for several hundred microseconds, is contributed by those Chla dag molecules which fail to transfer their excitation to a Car neighbour. A comparison of DELTA-1A(lambda; t) with the TmS signal of thylakoids shows differences which parallel those previously reported for the TmS spectra of trimers and aggregates of Chla/b-LHCII: the carotenoid peak at 510 nm is broader, and the Q-y depletion signal larger, in the difference spectrum of thylakoids. The absorption spectrum of Chla/c-LHCII show no signs of Chla-Chla excitonic interactions, since the Chla-contribution to the spectrum can be reproduced well by simply red-shifting (by about 200 cm-1) the Q bands and the Soret band in the absorption spectrum of an ethanolic solution of Chla, an observation consistent with the absence, reported in a recent study, of excitonic band in the absorption spectrum of an ethanolic solution of Chla, an observation consistent with the absence, reported in a recent study, of excitonic bands in the circular dichroism spectrum of Chla/c-LHCII.

DESCRIPTOR(S)- *ALGA; *ANALYSIS; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CHLOROPHYLL A; *CHLOROPHYLL C; *FLUORESCENCE-EXCITATION SPECTROMETRY; *FLUOROPHOTOMETRY: CB; *LABORATORY EQUIPMENT; *LHCII; *METHODOLOGY; *PHOTOMETRY: CB; *PHOTOSYNTHESIS; *PHOTOSYSTEM II; *PIGMENT-PIGMENT INTERACTIONS; *PLEUROCHLORIS MEIRINGENSIS; *SHIMADZU; *SHIMADZU MODEL UV-160A SPECTROPHOTOMETER; *SPECTROPHOTOMETRY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG 2 FLUORESCENCE SPECTROMETER; *THYLAKOIDS; *TRIPLET-MINUS-SINGLET SPECTROMETRY

1998

Phosphorylation-induced structural change in phospholamban and its mutants, detected by intrinsic fluorescence.

Li, M.; Cornea, R. L.; Autry, J. M.; Jones, L. R.; Thomas, D. D.

Biochemistry, VOL. 37, NO. 21, 1998, PP. 7869-7877

We have used intrinsic fluorescence to test the hypothesis that phosphorylation induces a conformational change in phospholamban (PLB), a regulatory protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB, which relieves inhibition of the cardiac Ca-ATPase, has been shown to decrease the mobility of PLB in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). In the present study, we found that this mobility shift depends on the acrylamide concentration in the gel, suggesting that phosphorylation increases the effective Stokes radius. To further characterize this structural change, we performed spectroscopic experiments.under the conditions of SDS-PAGE. CD indicated that phosphorylation at Ser-16 does not change PLB's secondary structure significantly. However, the fluorescence of Tyr-6 in the cytoplasmic domain of PLB changed significantly upon PLB phosphorylation: phosphorylation increased the fluorescence quantum yield and decreased the quenching efficiency by acrylamide, suggesting a local structural change that decreases the solvent accessibility of Tyr-6. A point mutation (L37A) in the transmembrane domain, which disrupts PLB pentamers and produces monomers in SDS -PAGE and in lipid bilayers, showed similar phosphorylation effects on fluorescence, indicating that subunit interactions within PLB are not crucial for the observed conformational change in SDS. When PLB was reconstituted into dioleoylphosphatidylcholine (DOPC) lipid bilayers, similar phosphorylation effects in fluorescence were observed, suggesting that PLB behaves similarly in response to phosphorylation in both detergent and lipid environments. We conclude that phosphorylation induces a structural change within the PLB protomer that decreases the solvent accessibility of Tyr-6. The similarity of this structural change in monomers and pentamers is consistent with models in which the PLB monomer is sufficient for the phosphorylation-dependent regulation of the Ca-ATPase.

DESCRIPTOR(S)- *ACRYLAMIDE GEL; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CALCIUM-ATPASE; *CIRCULAR DICHROISM; *DETECTION; *DETECTION METHOD; *DETECTION-LABELING TECHNIQUES; *DIOLEOYLPHOSPHATIDYLCHOLINE; *ELECTROPHORETIC TECHNIQUES; *FERGUSON PLOT; *INTRINSIC FLUORESCENCE; *JASCO J-710 SPECTROPOLARIMETER; *LABORATORY EQUIPMENT; *LIPID BILAYER; *MATHEMATICAL METHOD; *METHODOLOGICAL APPROACH; *METHODOLOGY; *MUTANTS; *PHOSPHOLAMBAN; *PHOSPHORYLATION; *RECONSTITUTION; *SARCOPLASMIC RETICULUM; *SDS-PAGE; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SPECTROSCOPIC TECHNIQUES; * SPEX-FLUOROLOG II SPECTROFLUOROMETER; *STRUCTURAL CHANGE

1998

Peptide models of local and long-range interactions in the molten globule state of human alpha-lactalbumin.

Demarest, S. J.; Fairman, R.; Raleigh, D. P.

Journal of Molecular Biology, VOL. 283, NO. 1, 1998, PP. 279-291

alpha-Lactalbumin, a small calcium-binding protein, forms an equilibrium molten globule state under a variety of conditions. A set of four peptides designed to probe the role of local interactions and the role of potential long-range interactions in stabilizing the molten globule of alpha-lactalbumin has been prepared. The first peptide consists of residues 20 through 36 of human alpha-lactalbumin and includes the entire B-helix. This peptide is unstructured in solution as judged by CD. The second peptide is derived from residues 101 through 120 and contains both the D and 3-10 helices. When this peptide is crosslinked via the native 28 to 111 disulfide to the B-helix peptide, a dramatic increase in helicity is observed. The crosslinked peptide is monomeric, as judged by analytical ultracentrifugation. The peptide binds 1-anilinonaphthalene-8-sulphonate (ANS) and the fluorescence emission maximum of the construct is consistent with partial solvent exposure of the tryptophan residues. The peptide corresponding to residues 101 to 120 adopts significant non-random structure in aqueous solution at low pH. Two hydrophobic clusters, one involving residues 101 through 104 and the other residues 115 through 119 have been identified and characterized by NMR. The hydrophobic cluster formed by residues 101 through 104 is still present in a smaller peptide containing only residues 101 to 111 of alpha-lactalbumin. The cluster also persists in 6 M urea. A non-native, pH-dependent interaction between the Y103 and H107 side-chains that was previously identified in the acid-denatured molten globule state was examined. This interaction was found to be more prevalent at low pH and may therefore be an example of a local interaction that stabilizes preferentially the acid-induced molten globule state.

DESCRIPTOR(S)- *ALPHA-LACTALBUMIN FRAGMENTS; *ANALYTICAL METHOD; *AVIV; *AVIV MODEL 62A CIRCULAR DICHROISM SPECTROMETER; *BECKMAN; *BECKMAN XL-A ANALYTICAL ULTRACENTRIFUGE; *BIOCHEMISTRY AND BIOPHYSICS; *CALCIUM-BINDING PROTEIN; *CENTRIFUGATION TECHNIQUES: CB; *CHARACTERIZATION; *CIRCULAR DICHROISM; *FLUORESCENCE MEASUREMENT; *FLUOROMETRY; *FOLDING; *HUMAN ALPHA-LACTALBUMIN; *ISA FLUOROLOG SPECTROMETER; *LABORATORY EQUIPMENT; *LOCAL INTERACTIONS; *LONG-RANGE INTERACTIONS; *METHODOLOGY; *MOLTEN GLOBULE STATE; *NMR; *PEPTIDE MODELS; *PROTEIN; *SEDIMENTATION EQUILIBRIUM ULTRACENTRIFUGATION; *SPECTROSCOPIC TECHNIQUES: CB; *VARIAN; *VARIAN INOVA 500 MEGAHERTZ SPECTROMETER; *VARIAN INOVA 600 MEGAHERTZ SPECTROMETER

1998

Metal-thiolate clusters in the C-terminal domain of human neuronal growth inhibitory factor (GIF).

Hasler, D. W.; Faller, P.; Vasak, M.

Biochemistry, VOL. 37, NO. 42, 1998, PP. 14966-14973

Neuronal growth inhibitory factor (GIF), a metallothionein-like protein (metallothionein-3), impairs the survival and neurite formation of cultured neurons. Native GIF contains 4 Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters. However, the cluster localization is not known. In this study, the metal-thiolate clusters formed with monovalent and divalent metal ions in the C-terminal domain of human GIF (GIF(32-68)) containing 11 cysteines were investigated. The cluster formation was followed by using electronic absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy, and in the case of Cu(I) complexes also by luminescence spectroscopy at 77 K. Spectroscopic studies on the Cu(I)-GIF(32-68) complexes showed the successive formation of two air-sensitive Cu-4S-8-9- and Cu-6S-11-clusters. With Zn(II) and Cd(II) ions, a well-defined M-4S-11-cluster is formed in which each metal ion is tetrahedrally coordinated by cysteine thiolates. In the 113Cd NMR spectra of 113Cd-4-GIF(32-68), recorded at 293 and 323 K, all four 113Cd resonances at 672.8, 620.9, 629.6, and 564.2 ppm were observed only at 323 K. Their detection at elevated temperature indicates a conformational flexibility of this domain. Evidence for the existence of a Cd-6-GIF(32-68) complex, containing two more weakly bound Cd(II) ions, was also obtained. The formation of this complex requires the transformation of some originally terminal thiolates of the Cd-4S-11-cluster to bridging thiolates, suggesting a more accessible cluster structure. Such properties of Cd-4-GIF(32-68) have not been observed with the Cd-4S-11-cluster in the isolated alpha-domain (amino acids 31-61) of metallothioneins. The significance of Cu- and Zn-clusters for the structure of native GIF is discussed.

DESCRIPTOR(S)- *ABI 433 A PEPTIDE SYNTHESIZER; *ANALYSIS; *ANALYTICAL METHOD; *ATOMIC ABSORPTION SPECTROMETRY; *BIOCHEMISTRY AND BIOPHYSICS; *BRUKER; *BRUKER AMX-600 SPECTROMETER; *CIRCULAR DICHROISM; *ELECTRONIC ABSORPTION SPECTROSCOPY; *ELECTROSPRAY MASS SPECTROMETRY; *HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; *HPLC; *HUMAN NEURONAL GROWTH INHIBITORY FACTOR CARBOXY-TERMINAL DOMAIN; *JASCO; *JASCO J-715 SPECTROPOLARIMETER; *LABORATORY EQUIPMENT; *LIQUID CHROMATOGRAPHY; *LUMINESCENCE SPECTROSCOPY; *MAGNETIC CIRCULAR DICHROISM SPECTROSCOPY; *MASS SPECTROMETRY: CB; *METAL-THIOLATE CLUSTERS; *METHODOLOGY; *NMR; *PURIFICATION; *PURIFICATION METHOD; *SEPHADEX G-10 COLUMN; *SPECTROSCOPIC TECHNIQUES: CB; *SPECTROSCOPY: CB; *SPEX FLUOROLOG SPECTROFLUOROMETER; *SYNTHESIS; *VARIAN; *VARIAN CARY 3 SPECTROMETER

1998

Membrane protein proteolysis assayed by fluorescence quenching: Assay of O-sialoglycoprotein endopeptidase.

Jiang, P.; Mellors, A.

Analytical Biochemistry, VOL. 259, NO. 1, 1998, PP. 8-15,

The assay of the O-sialoglycoprotein endopeptidase of Pasteurella haemolytica has previously used the cleavage of 125I-labeled glycophorin A, measured by SDS-PAGE, autoradiography, gel-slicing, and scintillation counting. A new assay is based on the increased fluorescence which results from proteolytic cleavage of a fluorescence-quenched micellar substrate, 4,4-difluor-5,7-di- methyl-4-bora-3-alpha,4-alpha-diaza-s-indacene-3-propionic acid conjugated to glycophorin A (BODIPY-FL-glycophorin A). Micellar association of glycophorin A molecules results in 97% fluorescence quenching despite a low molar ratio of BODIPY-FL-glycophorin A. Proteolysis of the membrane protein causes greatly enhanced fluorescence which is used for a rapid one-step proteolysis assay. Direct monitoring of proteolysis in microcuvettes, or routine assay in microtiter plates can be used. Reproducibility is higher than with the radiolabeled substrate and the K-m values for the two substrates are similar. The assay is suitable for the O-sialoglycoprotein endopeptidase activity of chromatographically purified enzyme or unpurified bacterial culture supernatants and can be used to monitor inhibition of the O-sialoglycoprotein endopeptidase by neutralizing antibodies. The O-sialoglycoprotein endopeptidase assay employing BODIPY-FL-glycophorin A provides a rapid and nonradioactive method for the assay of this highly specific enzyme.

DESCRIPTOR(S)- *METHODOLOGY; *FLUORESCENCE QUENCHING MEMBRANE PROTEIN PROTEOLYSIS ASSAY; *PASTEURELLA-HEMOLYTICA O-SIALOGLYCOPROTEIN ENDOPEPTIDASE; *ASSAY; *EC 3.4.24.57; *4,4-DIFLUOR-5,7-D- IMETHYL-4-BORA-3-ALPHA,4-ALPHA-DIAZA-S-INCANDACENE-3-PROPIONIC ACID-GLYCOPHORIN A CONJUGATE; *PROTEOLYSIS; *ENZYMOLOGY; *HITACHI F-2000 SPECTROPHOTOFLUORIMETER; * FLUOROLOG DM 3000 FLUORIMETER; *FL 500 FLUORESCENCE PLATE READER; *ANALYTICAL METHOD; *SPECTROSCOPIC TECHNIQUES; *EQUIPMENT; *SPEX INDUSTRIES INC.; *BIO-TEK INC.

1998

Mapping the serpin-proteinase complex using single cysteine variants of alpha-1-proteinase inhibitor Pittsburgh.

Stratikos, E.; Gettins, P. G. W.

Journal of Biological Chemistry, VOL. 273, NO. 25, 1998, PP. 15582-15589

To probe the covalent serpin-proteinase complex, we used wild-type and 4 new single cysteine variants (T85C, S121C, D159C, and D298C) of alpha-1-proteinase inhibitor Pittsburgh. Cysteines in each variant could be labeled both in native and proteinase-complexed alpha-1-proteinase inhibitors. Pre-reaction with 7-nitrobenz-2-oxa-1,3-diazole-chloride or fluorescein prevented complex formation only with the D298C variant. Label at Cys-121 greatly increased the stoichiometry of inhibition for thrombin and gave an emission spectrum that discriminated between native, cleaved, and proteinase-complexed serpin and between complexes with trypsin and thrombin, whereas fluorophore at residue 159 on helix F was almost insensitive to complex formation. Fluorescence resonance energy transfer measurements for covalent and non-covalent complexes were consistent with a location of the proteinase at the end of the serpin distal from the original location of the reactive center loop. Taken together, these findings are consistent with a serpin-proteinase complex in which the reactive center loop is fully inserted into beta-sheet A, and the proteinase is at the far end of the serpin from its initial site of docking with the reactive center loop close to, but not obscuring, residue 121.

DESCRIPTOR(S)- *ALPHA-1-PROTEINASE INHIBITOR PITTSBURGH; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES; *ANALYTICAL METHOD; *BETA-TRYPSIN LABELING SITE CHARACTERIZATION; *COVALENT COMPLEX FORMATION ABILITY ASSAY; *DETECTION METHOD; *DETECTION-LABELING TECHNIQUES; *DETECTION-LABELING TECHNIQUES: CB; *ENZYME INHIBITOR; *ENZYMOLOGY; *FLUORESCEIN; *FLUORESCENCE RESONANCE ENERGY TRANSFER MEASUREMENT; *GENETIC METHOD; *INHIBITION STOICHIOMETRY CALCULATION; *ISOLATION-PURIFICATION TECHNIQUES; *LABEL; *LABORATORY EQUIPMENT; *MAPPING; *MEASUREMENT METHOD; *METHODOLOGY; *MOLECULAR PROBES; *MUTAGENESIS TECHNIQUES; *PURIFICATION METHOD; *SERPIN-PROTEINASE COMPLEX; *SINGLE CYSTEINE VARIANTS; *SITE-DIRECTED MUTAGENESIS; *SPEX FLUOROLOG SCANNING FLUORIMETER; *TETRAMETHYLRHODAMINE ISOTHIOCYANATE; *TETRAMETHYLRHODAMINE ISOTHIOCYANATE LABELING; *THROMBIN; *TRYPSIN; *7-NITROBENZ-2-OXA-1,3-DIAZOLE-CHLORIDE

1998

Kinetics of dimerization and interactions of p13-suc1 with cyclin-dependent kinases.

Morris, M. C.; Heitz, F.; Divita, G.

Biochemistry, VOL. 37, NO. 40, 1998, PP. 14257-14266

The impact of p13-suc1 on the conformation and regulation of cyclin-dependent kinases (cdks) and cyclins was investigated by spectroscopic and rapid kinetic approaches. In the absence of phosphorylation on cdks, p13-suc1 formed stable complexes, mainly stabilized by hydrophobic interactions, specifically with cdk2 and cdc2. The presence of cyclin A, associated with cdk2 or cdc2, increased the stability of the interaction between cdk2 and p13-suc1 by a factor of 2. However, cyclin A did not modify the association rate of p13-suc1 to cdk2, but the dissociation rate, which was decreased 3-fold. Moreover, binding of p13-suc1 to cdk2 resulted in a 2-fold decrease in the release of nucleotide from cdk2, indicating that p13-suc1 induces a marked change in the structure of the nucleotide binding site of cdks. On the basis of the structure of cdk2/CksHs1 complex and on our kinetic results, we propose that the binding of Cks proteins to C-lobe of cdk2 is stabilized by the presence of cyclin A and that it may modify the orientation of the loop carrying residues 14 and 15 and their consequent access for dephosphorylation by cdc25 phosphatases. Finally, we have shown that dimerization of p13-suc1 in the presence of zinc abolishes its interaction with cdks, which suggests that the binding of p13-suc1 to cdk2 or cdk2/cyclin A may be regulated by dimerization of p13-suc1 in vivo.

DESCRIPTOR(S)- *ANALYSIS; *ANALYTICAL METHOD; *CHROMATOGRAPHIC TECHNIQUES; *CIRCULAR DICHROISM; *CYCLIN-DEPENDENT KINASES; *CYCLINS; *DIMERIZATION KINETICS; *ENZYMOLOGY; *EQUIPMENT; *FLUORESCENCE DETECTION; *FUNCTIONS; *MOLECULAR INTERACTIONS; *P-13-SUC; *SIZE EXCLUSION HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX II FLUOROLOG SPECTROFLUOROMETER; *STAINING-VISUALIZATION; *YEAST CELL CYCLE PROGRESSION; *YEASTS

1998

Kinetics of Competitive Adsorption of PEO chains with Different Molecular Weights,

Fu, Z. and Santore M. M.;

Macromolecules, Volume 81, (1998), October 4 Edition, in press.

 

1998

Intrastrand cross-linked actin between Gln-41 and Cys-374. III. Inhibition of motion and force generation with myosin.

Kim, E.; Bobkova, E.; Miller, C. J.; Orlova, A.; Hegyi, G.; Egelman, E. H.; Muhlrad, A.; Reisler, E.

Biochemistry, VOL. 37, NO. 51, 1998, PP. 17801-17809

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degree C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degree C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the K-d and K-m values were little affected by the crosslinking, and the V-max decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by apprx 35% while the relative force, as measured by using external load in these assays, was inhibited by apprx 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at apprx 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.

DESCRIPTOR(S)- *ACTIN; *ACTIVITY ASSAYS; *AMINO ACIDS; *ANALYSIS; *ANALYTICAL METHOD; *ATP ASSAY; *ATPASE; *BIOCHEMISTRY AND BIOPHYSICS; *ELECTRON MICROSCOPY; *EQUIPMENT; *FORCE GENERATION; *FUNCTIONS; *INTRASTRAND CROSS-LINKING; *LIGHT SCATTERING; *MICROSCOPY METHOD; *MICROSCOPY: CB; *MICROSCOPY: CT; *MOTION; *MUSCULAR SYSTEM; *MYOSIN; *RABBIT; *SKELETAL MUSCLE; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG SPECTROFLUOROMETER; *SPEX INDUSTRIES; *STRUCTURE; *TEMPERATURE EFFECTS

1998

Intrastrand cross-linked actin between Gln-41 and Cys-374. II. Properties of cross-linked oligomers.

Kim, E.; Phillips, M.; Hegyi, G.; Muhlrad, A.; Reisler, E.

Biochemistry, VOL. 37, NO. 51, 1998, PP. 17793-17800

Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s degree 20,w = 5.55 +- 0.22 S), trimers (s degree 20,w = 6.93 +- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment (S1) to cross-linked dimers in the presence of pyrenyl G-actin revealed about 2 orders of magnitude stronger binding of the first than that of the second S1 molecule to actin dimer. Under similar conditions the unpolymerized cross-linked actin species activated the MgATPase of S1 only severalfold compared to 70-fold activation by F-actin. The cross-linked dimers, trimers, and oligomers were polymerized into filaments by MgCl-2 faster than un-cross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than un-cross-linked actin filaments. Small amounts of cross-linked actin dimers nucleated S1-induced polymerization of actin, but the polymerization by S1 was inhibited for pure populations of cross-linked dimers, trimers, and oligomers. The cross-linked dimers did not decrease the kinetic difference between the polymerization of actin by S1 isozymes S1 (A1) and S1 (A2). According to electron microscopy evidence, cross-linked actin oligomers polymerized by S1 yielded much shorter arrowhead structures than the un-cross-linked actin. These results indicate the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by S1.

DESCRIPTOR(S)- *ACTIN; *ACTIN FILAMENTS; *ACTIVITY ASSAYS; *AMINO ACIDS; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *ATPASE; *BIOCHEMISTRY AND BIOPHYSICS; *CENTRIFUGATION TECHNIQUES: CB; *CROSS-LINKED ACTIN OLIGOMERS; *ELECTRON MICROSCOPY; *ENZYME ASSAY; *EQUIPMENT; *INTRASTRAND CROSS-LINKING; *LIGHT SCATTERING; *MICROSCOPY METHOD; *MICROSCOPY: CB; *MICROSCOPY: CT; *MOLECULAR CHARACTERISTICS; *MUSCULAR SYSTEM; *PROTEINS; *RABBIT; *SKELETAL MUSCLE; *SPEX FLUOROLOG SPECTROFLUOROMETER; *SPEX INDUSTRIES; *ULTRACENTRIFUGATION

1998

Intrastrand cross-linked actin between Gln-41 and Cys-374. I. Mapping of sites cross-linked in F-actin by N-(4-azido-2-nitrophenyl) putrescine.

Hegyi, G.; Mak, M.; Kim, E.; Elzinga, M.; Muhlrad, A.; Reisler, E.

Biochemistry, VOL. 37, NO. 51, 1998, PP. 17784-17792

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 ANG , constrains to that range the distance between the y-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin (Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836). The effect of cross-linking on the function of actin is described in the companion papers.

DESCRIPTOR(S)- *ACTINS; *AMINO ACID; *ANALYSIS; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CHROMATOGRAPHIC TECHNIQUES; *ELECTROPHORETIC TECHNIQUES; *EQUIPMENT; *F-ACTIN; *FLUORESCENT PROBES; *GEL FILTRATION CHROMATOGRAPHY; *INTRASTRAND CROSS-LINKING; *MASS SPECTROMETRY; *MOLECULAR CHARACTERISTICS; *MUSCULAR SYSTEM; *N-(AZIDO-2-NITROPHENYL) PUTRESCINE; *N-TERMINAL SEQUENCING; *PHOTO-CROSSLINKING; *PURIFICATION METHOD; *RABBIT; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SEQUENCING TECHNIQUES; *SKELETAL MUSCLE; *SPECTROFLUOROMETRY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG SPECTROFLUOROMETER; *SPEX INDUSTRIES; *STRUCTURAL MODELS; *SYNTHESIS-MODIFICATION TECHNIQUES; *SYNTHETIC METHOD

1998

Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain.

Kachel, K.; Ren, J.; Collier, R. J.; London, E.

Journal of Biological Chemistry, VOL. 273, NO. 36, 1998, PP. 22950-22956

The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices THS and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Rachel, IL, Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *ANTI-BODIPY ANTIBODIES; *BINDING; *BIOCHEMISTRY AND BIOPHYSICS; *BODIPY ASSAY; *BODIPY-IODOACETAMIDE; *CYSTEINE; *DETECTION METHOD; *DETECTION-LABELING TECHNIQUES; *DIPHTHERIA TOXIN; *FLUORESCENCE LABELING; *FLUORESCENCE MEASUREMENT; *HYDROPHOBIC HELICES; *ISOLATION METHOD; *ISOLATION-PURIFICATION TECHNIQUES: CB; *LABEL; *LABORATORY EQUIPMENT; *LIPID BILAYER; *MEASUREMENT METHOD; *MEMBRANE INCORPORATED T DOMAIN PREPARATION; *MEMBRANE-INSERTED DOMAIN; *MEMBRANES; *METHODOLOGY; *MOLECULAR PROBES; *MONOCHLOROBIMANE; *SPEX 212 FLUOROLOG SPECTROFLUOROMETER; *T DOMAIN; *TRANSMEMBRANE STATES

1998

Hydrophobic forces dominate the thermodynamic characteristics of UvrA-DNA damage interactions.

Zou, Y.; Bassett, H.; Walker, R.; Bishop, A.; Amin, S.; Geacintov, N. E.; Van Houten, B.

Journal of Molecular Biology, VOL. 281, NO. 1, 1998, PP. 107-119

The Escherichia coli DNA repair proteins UvrA, UvrB and UvrC work together to recognize and incise DNA damage during the process of nucleotide excision repair (NER). To gain an understanding of the damage recognition properties of UvrA, we have used fluorescence spectroscopy to study the thermodynamics of its interaction with a defined DNA substrate containing a benzo(a)pyrene diol epoxide (BPDE) adduct. Oligonucleotides containing a single site-specifically modified N-2-guanine (+)-trans-, (-)-trans-, (+)-cis-, or (-)-cis-BPDE adducts were ligated into 50-base-pair DNA fragments. All four stereoisomers of DNA-BPDE adducts show an excitation maximum at 350 nm and an emission maximum around 380 to 385 run. Binding of UvrA to the BPDE-DNA adducts results in a five to sevenfold fluorescence enhancement. Titration of the BPDE-adducted DNA with UvrA was used to generate binding isotherms. The equilibrium dissociation constants for UvrA binding to (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis- BPDE adduct were: 7.4 +- 1.9, 15.8 +- 5.4, 11.3 +- 2.7 and 22.4 +- 2.0 nM, respectively. There was a large negative change in heat capacity DELTA-p,obs-o, (-3.3 kcal mol-1 K-1) accompanied by a relatively unchanged DELTA-G-obs-o with temperature. Furthermore, varying the concentration of KCl showed that the number of ions released upon formation of UvrA-DNA complex is about 3.4, a relatively small value compared to the contact size of UvrA with the substrate. These data suggest that hydrophobic interactions are an important driving force for UvrA binding to BPDE-damaged DNA.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL GEL FILTRATION; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *DNA BINDING; *DNA DAMAGE; *DNA REPAIR PROTEIN; *ESCHERICHIA-COLI UVRA PROTEIN; *FILTRATION TECHNIQUES; *FLUORESCENCE SPECTROSCOPY; *HYDROPHOBIC FORCES; *LABORATORY EQUIPMENT; *METHODOLOGY; *NUCLEOTIDE EXCISION REPAIR; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX; *SPEX FLUOROLOG FLUOROMETER; *THERMODYNAMIC CHARACTERISTICS; *UVRA-DNA COMPLEX

1998

Hydrophobic core substitutions in calbindin D-9k: Effects on stability and structure.

Julenius, K.; Thulin, E.; Linse, S.; Finn, B. E.

Biochemistry, VOL. 37, NO. 25, 1998, PP. 8915-8925

The effects of hydrophobic core mutations on the stability and structure of the four-helix calcium-binding protein, calbindin D-9k, have been investigated. Eleven mutations involving eight residues distributed within the hydrophobic core of calbindin D-9k were examined. Stabilities were measured by denaturant and thermal induced unfolding monitored by circular dichroism spectroscopy. The mutations were found to exert large effects on the stability with midpoints in the urea induced unfolding varying from 1.8 M for Leu23 fwdarw Gly up to 6.6 M for Va170 dag AR Leu and free energies of unfolding in the absence of denaturant ranging from 6.6 to 27.4 kJ/mol for the Phe66 fwdarw Ala mutant and the wild-type, respectively. A significant correlation was found between the difference in free energy of unfolding (DELTA-DELTA-G-NU) and the change in the surface area of the side chain caused by the mutation, in agreement with other studies. Notably, both increases and decreases in side-chain surface area caused quantitatively equivalent effects on the stability. In other words, a correlation between the absolute value of the change in the surface of the side chain and DELTA-DELTA-G-NU was observed with a value of approximately 0.14 kJ M-1 ANG -2. The generality of this observation is discussed. Significant effects on the cooperativity of the unfolding reaction were also observed. However, a correlation between the cooperativity and DELTA-DELTA-G-NU, which has been reported in other systems as an indication of effects of mutations on the unfolded state, was not observed for calbindin D-9k. Despite the large effects on DELTA-DELTA-G-NU and cooperativity, the structures of the mutants in the native form remained intact as indicated by circular dichroism, NMR, and fluorescence measurements. The structural response to calcium-binding was also conserved. The following paper in this issue (Kragelund, B. B., et al. (1998) Biochemistry 37, 8926-8937) examines the effects of these mutations on the calcium binding properties of calbindin D-9k.

DESCRIPTOR(S)- *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CALBINDIN D-9K; *CALCIUM BINDING PROPERTY; *CIRCULAR DICHROISM; *EQUIPMENT; *FLUORESCENCE SPECTRA; *GE OMEGA NMR SPECTROMETER; *HYDROPHOBIC CORE SUBSTITUTIONS; *JASCO; *JASCO J-720 SPECTROPOLARIMETER; *METHODOLOGY; *MONITORING METHOD; *MUTAGENESIS TECHNIQUES; *NMR; *PROTEIN FOLDING; *PROTEIN MUTAGENESIS; *PROTEIN STABILITY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG FLUORESCENCE SPECTROMETER; *STRUCTURE

1998

Hybridization of peptide nucleic acid.

Ratilainen, T.; Holmen, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.; Norden, B.

Biochemistry, VOL. 37, NO. 35, 1998, PP. 12331-12342

The thermodynamics of hybridization and the conformations of decameric mixed purine-pyrimidine sequence PNA/PNA, PNA/DNA, and DNA/DNA duplexes have been studied using fluorescence energy transfer (FET), absorption hypochromicity (ABS), isothermal titration calorimetry (ITC), and circular dichroism (CD) techniques. The interchromophoric distances determined in the FET experiments on fluorescein- and rhodamine-labeled duplexes indicate that the solution structures of the duplexes are extended helices in agreement with available NMR (PNA/DNA) and crystal X-ray data (PNA/PNA). The melting thermodynamics of the duplexes was studied with both FET and ABS. The thermodynamic parameters obtained with ABS are in good agreement with the parameters from calorimetric measurements while FET detection of duplex melting gives in most cases more favorable free energies of hybridization. This discrepancy between FET and ABS detection is ascribed to the conjugated dyes which affect the stability of the duplexes substantially. Especially, the dianionic fluorescein attached via a flexible linker either to PNA or to DNA seems to be involved in an attractive interaction with the opposite dicationic lysine when hybridized to a PNA strand. This interaction leads to an increased thermal stability as manifested as a 3-4 degree C increase of the melting temperature. For the PNA/DNA duplex where fluorescein is attached to the PNA strand, a large destabilization (DELTA-T-m = - 12 degree C) occurs relative to the unlabeled duplex, probably originating from electrostatic repulsion between the fluorescein and the negatively charged DNA backbone. In the case of the PNA/PNA duplex, the sense of helicity of the duplex is reversed upon conjugation of fluorescein via a flexible linker arm, but not when the fluorescein is attached without a linker to the PNA.

DESCRIPTOR(S)- *ABSORPTION HYPOCHROMICITY; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CARY; *CARY 4 SPECTROPHOTOMETER; *CIRCULAR DICHROISM; *DNA-DNA DUPLEX; *EQUIPMENT; *FLUORESCENCE DETECTION; *FLUORESCENCE ENERGY TRANSFER; *HYBRIDIZATION; *ISOTHERMAL TITRATION CALORIMETRY; *JASCO; *JASCO 700 SPECTROPOLARIMETER; *METHODOLOGY; *MICROCAL; *MICROCAL ISOTHERMAL TITRATION CALORIMETRY MC-2 SYSTEM; *NMR SPECTROSCOPY; *PEPTIDE NUCLEIC ACID; *PEPTIDE NUCLEIC ACID-DNA DUPLEX; *PEPTIDE NUCLEIC ACID-PEPTIDE NUCLEIC ACID DUPLEX; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX; *SPEX FLUOROLOG TAU-2 APPARATUS; *THERMODYNAMICS; *X-RAY ANALYSIS; *X-RAY CRYSTALLOGRAPHY

1998

From coiled to small globular proteins: Design of a native-like three-helix bundle.

Bryson, J. W.; Desjarlais, J. R.; Handel, T. M.; Degrado, W. F.

Protein Science, VOL. 7, NO. 6, 1998, PP.1404-1414

A monomolecular native-like three-helix bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three-helix bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine-induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native-like state was achieved through the introduction of: (1) helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES; *ANALYTICAL METHOD; *BECKMAN XLA ANALYTICAL ULTRACENTRIFUGE; *BIOCHEMISTRY AND BIOPHYSICS; *BRUKER AMX-600 SPECTROMETER; *CENTRIFUGATION TECHNIQUES; *CHROMATOGRAPHIC TECHNIQUES; *EQUIPMENT; *FLUORESCENCE SPECTROSCOPY; *GLOBULAR PROTEINS; *HYDROPHOBIC CORE PACKING; *ISOLATION METHOD; *METHODOLOGY; *MILLIGEN 9050 AUTOMATED PEPTIDE SYNTHESIZER; *NMR; *PEPTIDE SYNTHESIS; *PROTEIN DESIGN; *PROTEIN FOLDING; *SEDIMENTATION EQUILIBRIUM; *SIZE EXCLUSION CHROMATOGRAPHY; *SPECTROSCOPIC TECHNIQUES; *SPEX FLUOROLOG FLUORIMETER; *SYNTHETIC METHOD; *SYNTHETIC TECHNIQUES; *THERMODYNAMICS; *THREE-HELIX BUNDLE

1998

Flow cytometric characterization and classification of multiple dual-color fluorescent microspheres using fluorescence lifetime.

Keij, J. F.; Steinkamp, J. A.

Cytometry, VOL. 33, NO. 3, 1998, PP. 318-323

FlowMetrix (Luminex, Austin, TX) microspheres were recently introduced as a platform for bead-based assays involving antibodies, enzymes, toxins, and nucleic acids. The procedure involves classification of the microspheres by their orange and red fluorescence and quantitation of the BODIPY-tagged biological probes by their green fluorescence. In an attempt to increase the number of fluorochromes available for the biological probes, we explored the possibility of using excited singlet state lifetime as an alternative to one of the fluorochromes. For a set of 20 dual-color microspheres the excited singlet state lifetimes were measured using the total emissions ( gt 515 nm), the orange emissions (515-600 nm), and the red emissions ( gt 665 nm). The microspheres could not all be resolved in bivariates of fluorescence intensity versus excited singlet state lifetime. However, 13 of the microspheres could be resolved using the total emissions and lifetime. Although this result required both fluorochromes, the merits and limitations of this approach to other systems are briefly discussed.

DESCRIPTOR(S)- *CELL ANALYTICAL METHOD; *CELL BIOLOGY; *CYTOPHOTOMETRY: CT; *EQUIPMENT; *FLOW CYTOMETRY; *FLOWMETRIX FLUORESCENT MICROSPHERES; *FLUOROCHROMES; * FLUOROLOG FLUOROMETER; *METHODOLOGY; *ORANGE FLUORESCENCE; *RED FLUORESCENCE

1998

Evidence for hydrophobic interaction between galanin and the Ga1R1 galanin receptor and Ga1R1-mediated ligand internalization: Flourescent probing with a fluorescein-galanin.

Wang, S.; Clemmons, A.; Strader, C.; Bayne, M.

Biochemistry, VOL. 37, NO. 26, 1998, PP. 9528-9535

Galanin is a neuropeptide that activates specific receptors to modulate several physiological functions including food intake, nociception, and learning and memory. The molecular nature of the interaction between galanin and its receptors and the fate of the galanin/receptor complex after the binding event are not understood. A fluorescein-N-galanin (F-Gal) was generated to measure the interaction between galanin and rat GalR1 galanin receptor (rGalR1) and rGalR1-mediated ligand internalization using flow cytometry in transfected Chinese hamster ovary (CHO) cells. Like galanin, F-Gal bound rGalR1 with high affinity and stimulated intracellular signaling events. Fluorescence quenching by soluble KI of rGalR1-bound F-Gal revealed a highly protected environment around the fluorescein, suggesting that the N-terminal portion of galanin, which constitutes the binding site of galanin for the receptor, binds to a protected hydrophobic binding pocket within the receptor. Exposure to F-Gal stimulated rapid (t-1/2 apprx 10 min) and extensive (78%) internalization of surface F-Gal into rGalR1/CHO cells at 37 degree C but not at 0 degree C. In addition, the internalization did not occur in parental CHO cells at either 0 or 37 degree C and was inhibited by addition of 0.25 M sucrose in the medium, indicating a GalR1-mediated energy-requiring endocytic process. These results revealed a hydrophobic interaction between galanin and the GalR1 receptor, which is in contrast to those of other G protein-coupled receptors that mainly require hydrophilic interaction with their peptide ligands near or outside the plasma membrane surface, and illustrated that the initial binding interaction is followed by rapid cellular internalization of the agonist/GalR1 complex.

DESCRIPTOR(S)- *ACTIVITY ASSAY; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BECTON DICKINSON IMMUNOCYTOMETRY SYSTEMS, INC.; *BIOCHEMISTRY AND BIOPHYSICS; *BIOTINYL-RAT GALANIN; *CAMP; *CHO CELL LINE; *CYCLIC AMP; *CYCLIC AMP SCINTILLATION PROXIMITY ASSAY; *CYCLIC AMP SCINTILLATION PROXIMITY ASSAY KIT; *DETECTION METHOD; *DETECTION-LABELING TECHNIQUES; *DUPONT; *FACSCAN FLOW CYTOMETER; *FLOW CYTOMETRY; *FLUORESCEIN; *FLUORESCEIN-N-GALANIN; *FLUORESCENCE QUENCHING; *FLUORESCENCE SPECTROSCOPY; *FLUORESCENT PROBE; *FLUORESCENT PROBING; *GALANIN; *GALANIN RECEPTOR; *GALANIN-GALR1 RECEPTOR HYDROPHOBIC INTERACTION; *GALANIN-RECEPTOR COMPLEX FATE; *GALR1; *INTRACELLULAR CYCLIC AMP PRODUCTION; *LABORATORY EQUIPMENT; *MEASUREMENT; *METHODOLOGY; *MOLECULAR PROBES, INC.; *NEUROPEPTIDE; *PENINSULA LABORATORIES; *PORCINE IODINE-125 GALANIN; *RADIOLIGAND BINDING ASSAY; *RAT GALANIN; *SPECTROSCOPIC METHOD; *SPECTROSCOPIC TECHNIQUES: CB; *SPECTROSCOPIC TECHNIQUES: CT; *SPEX FLUOROLOG-2 FLUOROMETER; *SPEX INDUSTRIES INC.
BIOSIS Concept Code(s)- 02506; 10054; 10064; 10508

1998

Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F-1-ATPase catalytic sites measured using specific tryptophan probes.

Weber, J.; Senior, A. E.

Journal of Biological Chemistry, VOL. 273, NO. 50, 1998, PP. 33210-33215

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F-1-ATPase was measured in the presence of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in catalytic sites were used to determine nucleotide binding parameters. Azide brought about small decreases in K-d(MgATP) and K-d(MgADP). Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state with three catalytic site-bound MgATP and zero bound MgADP. These results rule out the idea that azide inhibits by "trapping" MgADP. Rather, azide blocks the step at which signal transmission between catalytic sites promotes multisite hydrolysis. Aurovertin bound with stoichiometry of 1.8 (mol/mol of F-1) and allowed significant residual turnover. Cycling of the aurovertin-free beta-subunit catalytic site through three normal conformations was indicated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that it acts to slow the switch of catalytic site affinities ("binding change step") subsequent to MgATP hydrolysis. Dicyclohexylcarbodiimide shifted the ratio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affecting K-d(MgATP) values. Like azide, it also appears to affect activity at the step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolsis.

DESCRIPTOR(S)- *ACTIVITY ASSAYS; *AMINCO-BOWMAN; *AMINCO-BOWMAN 2 SPECTROFLUOROMETER; *ANALYTICAL METHOD; *ATPASE INHIBITION ASSAY; *AUROVERTIN; *AZIDE; *DICYCLOHEXYLCARBODIIMIDE; *ENZYMOLOGY; *EQUILIBRIUM NUCLEOTIDE BINDING; *F-1-ATPASE; *LABORATORY EQUIPMENT; *MEASUREMENT; *METHODOLOGY; *NUCLEOTIDE BINDING; *PHOTOMETRY: CB; *SIGMA; *SPECTROFLUOROMETRY; *SPEX FLUOROLOG 2 SPECTROFLUOROMETER; *TRYPTOPHAN PROBES

1998

Deconvolution of the fluorescence emission spectrum of human antithrombin and identification of the tryptophan residues that are responsive to heparin binding.

Meagher, J. L.; Beechem, J. M.; Olson, S. T.; Gettins, P. G. W.

Journal of Biological Chemistry, VOL. 273, NO. 36, 1998, PP. 23283-23289

Heparin causes an allosterically transmitted conformational change in the reactive center loop of antithrombin and a 40% enhancement of tryptophan fluorescence. We have expressed four human antithrombins containing single Trp fwdarw Phe mutations and determined that the fluorescence of antithrombin is a linear combination of the four tryptophans. The contributions to the spectrum of native antithrombin at 340 nm were 8% for Trp-49, 10% for Trp-189, 19% for Trp-225, and 63% for Trp-307. Trp-225 and Trp-307 accounted for the majority of the heparin-induced fluorescence enhancement, contributing 37 and 36%, respectively. Trp-49 and Trp-225 underwent spectral shifts of 15 nm to blue and 5 nm to red, respectively, in the antithrombin-heparin complex. The blue shift for Trp-49 is consistent with partial burial by contact with heparin, whereas the red shift for Trp225 and large enhancement probably result from increased solvent access upon heparin-induced displacement of the contact residue Ser-380. The enhancement for Trp-307 may result from the heparin-induced movement of helix H seen in the crystal structure. The time-resolved fluorescence properties of individual tryptophans of wild-type antithrombin were also determined using the four variants and showed that Trp-225 and Trp-307 experienced the largest change in lifetime upon heparin binding, providing support for the steady-state fluorescence deconvolution.

DESCRIPTOR(S)- *AFFINITY CHROMATOGRAPHY; *ANALYSIS; *ANALYTICAL METHOD; *ANION EXCHANGE CHROMATOGRAPHY; *BCA PROTEIN ASSAY REAGENT KIT; *BIOCHEMISTRY AND BIOPHYSICS; *CDNA; *CIRCULAR DICHROISM; *COHERENT ANTARES; *COLUMN CHROMATOGRAPHY; *COMPLEMENTARY DNA; *EXPRESSION; *FLUORESCENCE EMISSION SPECTRUM; *GEL ELECTROPHORESIS; *HELIX H MOVEMENT; *HEPARIN BINDING; *HUMAN ANTITHROMBIN; *JASCO; *JASCO 710 SPECTROPOLARIMETER; *LABORATORY EQUIPMENT; *LIQUID CHROMATOGRAPHY; *METHODOLOGY; *MUTAGENESIS; *MUTANT; *MUTATION; *NEODYMIUM-YAG LASER; *PIERCE; *PROTEIN ENGINEERING; *PURIFICATION METHOD; *SDS-PAGE; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SITE-DIRECTED MUTAGENESIS; *SIZE-EXCLUSION CHROMATOGRAPHY; *SLM-AMINCO 8000 SPECTROFLUOROMETER; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX; *SPEX FLUOROLOG 2 SPECTROFLUOROMETER; *STEADY-STATE FLUORESCENCE DECONVOLUTION

1998

Competitive Adsorption of PEO Chains with and without Charged End Groups.

Fu, Z. and Santore, M. M.;

Langmuir, 14, (1998), pp. 4300 - 4307.

 

1998

Characterization of Polystyrene Latex Surfaces by the Adsorption of Rhodamine 6G.

Mubarekyan, E. and Santore, M. M.;

Langmuir, 14, (1998), pp. 1597 - 1608.

 

1998

Binding of terbium and cisplatin to C13* human ovarian cancer cells using time-resolved terbium luminescence.

Canada, R. G.; Paltoo, D. N.

Biochimica et Biophysica Acta, VOL. 1448, NO. 1, 1998, PP. 85-98

Terbium (Tb-3+) has been shown to increase the cellular accumulation and cytotoxicity of cisplatin in cisplatin-resistant human breast and ovarian cancer cells. Time-resolved Tb-3+ luminescence was used to describe the binding of cisplatin to cisplatin-resistant C13* cells. A high-affinity Tb-3+ binding site was identified in the plasma membrane of the C13* cells (n = 105 +- 2 fmol/cell and K-d = 36.3 +- 5.2 mu-M). The binding of Tb-3+ is suggested to occur through a cation-tau interaction with tryptophan residues in the plasma membrane, resulting in an enhancement of the intensity and lifetime of Tb-3+. Stern-Volmer quenching analysis revealed that the Tb-3+ binding site is not readily accessible to the aqueous environment. The quenching of the Tb-3+-C13* intensity by cisplatin occurred by static quenching processes, involving both a direct electron-exchange interaction as well as an indirect dipole-dipole resonant energy transfer mechanism. Formation of the Tb-3+-C13*cisplatin complex does not interfere with the high-affinity binding of Tb-3+; cisplatin and Tb-3+ bind within 5 to 10 ANG of each other. A specific terbium/cisplatin binding protein is suggested to play a role in the cellular accumulation and cytotoxicity of cisplatin. Therefore, the transport of cisplatin across the plasma membrane must also involve a facilitated diffusion process. Our results indicate that the binding of Tb-3+ to the plasma membrane may be potentially useful in the reversal of cisplatin resistance.

DESCRIPTOR(S)- *ALDRICH CHEMICAL CO.; *ANALYTICAL METHOD; *ANTINEOPLASTIC-DRUG; *CISPLATIN; *C13 CELL LINE; *DRUG RESISTANCE; *FLUOROMETRY: CT; *HUMAN OVARIAN CANCER CELLS; *LABORATORY EQUIPMENT; *MATHEMATICAL METHOD; *METHODOLOGY; *PHARMACOLOGY; *REVERSAL; *SPEX INDUSTRIES; *SPEX INDUSTRIES FLUOROLOG-2 MODEL F212I SPECTROFLUOROMETER; *STERN-VOLMER QUENCHING ANALYSIS; *TERBIUM; *TIME-RESOLVED TERBIUM LUMINESCENCE; *TUMOR BIOLOGY

1998

Analysis of binding and membrane destabilization of phospholipid membranes by surfactant apoprotein B.

Chang, R.; Nir, S.; Poulain, F. R.

Biochimica et Biophysica Acta, VOL. 1371, NO. 2, 1998, PP. 254-264

To further elucidate the nature of the molecular interactions of surfactant apoprotein B (SP-B) with phospholipid (PL) membranes, we studied the binding of SP-B to PL membranes and the lipid-dependency of its subsequent effects on leakage and fusion of membranes. SP-B binding to membranes was studied by labeling the protein with the fluorophore 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) and measuring the fluorescence of the labeled protein in the presence of varying amounts of dipalmitoylphosphafidylcholine-egg phosphatidylclycerol (DPPC-eggPG; 7-3). Leakage of contents from liposomes made of DPPC and varying molar fraction of egg phosphatidylcholine (eggPC) or eggPG was assessed by measuring the fluorescence of entrapped water-soluble probes ANTS and DPX. Fusion of membranes was assessed by measuring the fluorescence of membrane-bound NBD-phosphatidylethanolamine (NBD-PE) and rhodamine-PE (RHOPE). We found that SP-B bound to PL membranes with high affinity and appeared to irreversibly cluster at the membrane surface, leading to graded release of the vesicle contents and eventually fusion of the membranes with increasing protein-lipid ratios. All lipid mixtures tested were susceptible to the membrane disruptive effects of SP-B, but DPPC-eggPG membranes displayed a biphasic response to increasing molar fractions of eggPG, whereas increasing fractions of eggPC elicited a monotonic response.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES; *ANALYTICAL METHOD; *AVANTI POLAR LIPIDS; *BINDING; *BIOCHEMISTRY AND BIOPHYSICS; *DETECTION-LABELING TECHNIQUES: CB; *DIPALMITOYLPHOSPHATIDYLCHOLINE-EGG PHOSPHATIDYLGLYCEROL; *DOG; *ELECTROPHORETIC TECHNIQUES: CT; *FLUORESCENCE SPECTROSCOPY; * FLUOROLOG 2 SPECTROFLUOROMETER; *FLUOROPHORE; *HUMAN; *LABELING METHOD; *LABORATORY EQUIPMENT; *LIPID-PROTEIN INTERACTIONS; *LIPOSOMES; *MEASUREMENT; *MEMBRANE DESTABILIZATION; *MEMBRANE PERMEABILITY; *MEMBRANES; *METHODOLOGY; *PHOSPHOLIPID MEMBRANES; *PROBE; *RHODAMINE-PHOSPHATIDYLETHANOLAMINE; *SDS-PAGE; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX INDUSTRIES; *SURFACTANT APOPROTEIN B; *SURFACTANT PROTEIN-B LABELING; *7-NITRO-2,1,3-BENZOXADIAZOL-4-YL; *7-NITRO-2,1,3-BENZOXADIAZOL-4-YL-PHOSPHATIDYLETHANOLAMINE

1998

A test of the relationship between sequence and structure in proteins: Excision of the heme binding site in apocytochrome b-5.

Constans, A. J.; Mayer, M. R.; Sukits, S. F.; Lecomte, J. T. J.

Protein Science, VOL. 7, NO. 9, 1998, PP. 1983-1993

The water-soluble domain of rat hepatic holocytochrome b-5 is an alpha-beta protein containing elements of secondary structure in the sequence beta-1-alpha-1-bet- a-4-beta-3-alpha-2-alpha-3-beta-4-alpha-5-beta-2-alpha-6. The heme group is enclosed by four helices, alpha-2, alpha-3, alpha-4, and alpha-5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta-1-alpha-1-beta-4-LAMBDA-beta-2-alpha-6 was prepared, where A is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.

DESCRIPTOR(S)- *AMINO ACID SEQUENCE; *ANALYSIS; *ANALYTICAL METHOD; *APOCYTOCHROME B-5; *AVIV 62DS CD SPECTROPOLARIMETER; *BRUKER; *BRUKER AMX2-500 SPECTROMETER; *BRUKER DRX-600 SPECTROMETER; *CHROMATOGRAPHIC TECHNIQUES; *CIRCULAR DICHROISM SPECTROSCOPY; *COMPUTER SOFTWARE; *ENZYMOLOGY; *ESCHERICHIA COLI; *EXPRESSION SYSTEM; *FAST PROTEIN LIQUID CHROMATOGRAPHY; *FLUORESCENCE EMISSION SPECTROSCOPY; *LABORATORY EQUIPMENT; *MALDI MASS SPECTROMETRY; *MASS SPECTROMETRY: CB; *MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS SPECTROMETRY; *METHODOLOGY; *NMR; *PHARMACIA; *PHARMACIA-LKB FPLC SYSTEM; *PURIFICATION METHOD; *SEPHAROSE COLUMN CHROMATOGRAPHY; *SEQUENCE-STRUCTURE RELATIONSHIP; *SIZE-EXCLUSION CHROMATOGRAPHY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROLOG 1681 FLUORIMETER; *STRAIN-BL21; *SUPEROSE 12 COLUMN; *THERMAL DENATURATION; *X-PLOR