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

2002

Visualization of proteins in acrylamide gels using ultraviolet illumination.

Kazmin, Dmitri; Edwards, Robert A.; Larson, Eric; Starkey, Jean; Turner, Raymond J.

Analytical Biochemistry, VOL. 301, NO. 1, February 1, 2002, PP. 91-96

Proteins in polyacrylamide gels can be rapidly visualized by soaking in trichloroacetic acid or chloroform followed by illumination with UV light. The UV-light-driven reaction of tryptophan in the presence of trichlorocompounds yields products that emit sufficiently in the visible region to identify the location of the protein bands on the gel. This method can be used to rapidly identify protein bands on a gel in less than 20 min. On thin polyacrylamide gels, 1.0 mug of protein can easily be detected for proteins with typical tryptophan percentages.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *chloroform; *proteins --detection; *trichloroacetic acid; *tryptophan; *acrylamide gels --laboratory equipment; *electrophoresis apparatus --laboratory equipment; *electrophoresis apparatus --Owl Scientific; *gel cassettes --laboratory equipment; *protein visualization --imaging method; *protein visualization --Imaging Techniques; *Bio-Rad Gel Doc system --laboratory equipment; *Bio-Rad Gel Doc system --Bio-Rad; * Fluorolog-3 fluorometer --laboratory equipment; * Fluorolog-3 fluorometer --ISA Jobin-Yvon Spex; *Mini Protean II system --laboratory equipment; *Mini Protean II system --Bio-Rad; *UltraLum transilluminator --laboratory equipment; *UV illumination --imaging method; *UV illumination --Imaging Techniques; *UV transilluminator --laboratory equipment; *protein bands; *UV light

2002

Tryptophan fluorescence studies of melanotropins in the amphiphile-water interface of reversed micelles.

Ito, Amando Siuiti; Carneiro Fernandes Souto, Ana Lucia

European Biophysics Journal, VOL. 29, NO. 1, 2002, PP. 38-47

We report studies on the interaction of alpha-melanocyte stimulating hormone (alpha-MSH) and a synthetic analogue (MSH-I) with reverse micelles prepared from the amphiphilic sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. The tripeptide lysyl-tryptophyllysine and the isolated amino acid tryptophan were also investigated as simpler compounds interacting with the micelles. Tryptophan fluorescence parameters (spectral position of emission band, anisotropy, and lifetime decay) demonstrated that in the presence of reverse micelles the environment around the fluorophore is less polar and more rigid than bulk water. Those parameters are sensitive to the changes induced in the micelles by the presence of water. In large micelles having a water/amphiphile molar ratio above 10, the modifications detected by fluorescence are small and the location of the fluorophore is not affected by a further increase in the concentration of the bulk water. The results, with additional support from quenching experiments, indicated that the different compounds occupy different positions in the large reverse micelles, but in any case they are in the interface region, without dispersing into the bulk water. From decay associated spectra, conformations were identified showing different degrees of tryptophan exposition to polar and nonpolar local environments. The conformation related to the long lifetime has its tryptophan more exposed to water while that associated to the intermediate lifetime has that residue stabilized in nonpolar media. The native hormone alpha-MSH and the analogue MSH-I present similar conformations in dry micelles. However, in buffer and in the large hydrated micelles, differences in conformations are evident, and could be related to the different physiological activity of the peptides.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Endocrine System (Chemical Coordination and Homeostasis); *Methods and Techniques; *alpha-melanocyte stimulating hormone alpha-MSH --analysis; *alpha-melanocyte stimulating hormone alpha-MSH --quantitative structure-activity relationships; *lysyl-tryptophyl-lysine --analysis; *lysyl-tryptophyl-lysine --quantitative structure-activity relationships; *lysyl-tryptophyl-lysine --tripeptide; *melanotropins --analysis; *melanotropins --tryptophan fluorescence; *reversed micelle --analysis; *reversed micelle --water-amphiphile molar ratio; *MSH-I --analysis; *MSH-I --quantitative structure-activity relationships; *MSH-I --synthetic analogue; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *optical absorption spectroscopy --analytical method; *optical absorption spectroscopy --Spectrum Analysis Techniques; * Fluorolog 3 Jobin Yvon-Spex spectrometer --equipment; *HP 8452 A diode array spectrometer --equipment

2002

Topography of helices 5-7 in membrane-inserted diphtheria toxin T domain. Identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin.

London, Erwin; Rosconi, Michael P.

Journal of Biological Chemistry, VOL. 277, NO. 19, May 10, 2002, PP. 16517-16527

The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state). We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis. After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored. It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state. Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9. The boundaries of deeply inserting sequences were also identified. One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7. This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink. Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state. Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin. However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure. How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *Corynebacterium diphtheriae (Irregular Nonsporing Gram-Positive Rods); *Bacteria; *Eubacteria; *Microorganisms; *membrane-inserted diphtheria toxin T domain --helix 5 topography; *membrane-inserted diphtheria toxin T domain --helix 6 topography; *membrane-inserted diphtheria toxin T domain --helix 7 topography; *membrane-inserted T domain; *transmembrane hairpin --hydrophobic sequences; *T domain molecules; *site-directed mutagenesis --mutagenesis/deletion; *site-directed mutagenesis --protein engineering; *Spex 212 Fluorolog spectrophotometer --Spectrum Analysis Techniques; *hydrophobic helices; *planar bilayer studies; *translocation

2002

The PDZ1 domain of SAP90. Characterization of structure and binding.

Mierke, Dale F.; Marshall, John; Blackman, Scott M.; Garcia, Elizabeth P.; Mehta, Sunil; Pellegrini, Maria; Piserchio, Andrea

Journal of Biological Chemistry, VOL. 277, NO. 9, March 1, 2002, PP. 6967-6973

The structural features of the PDZ1 domain of the synapse-associated protein SAP90 have been characterized by NMR. A comparison with the structures of the PDZ2 and PDZ3 domains of SAP90 illustrates significant differences, which may account for the unique binding properties of these homologous domains. Within the postsynaptic density, SAP90 functions as a molecular scaffold with a number of the protein-protein interactions mediated through the PDZ1 domain. Here, using fluorescence anisotropy and NMR chemical shift analysis, we have characterized the association of PDZ1 to the C-terminal peptides of the GluR6 subunit of the kainate receptor, voltage-gated K+ channel Kv1.4, and microtubule-associate protein CRIPT, all of which are known to associate with SAP90. The latter two, which possess the consensus sequence for binding to PDZ domains (T/S-X-V-oh), have low micromolar binding affinities (1.5-15 muM). The C terminus of GluR6, RLPGKETMA-oh, lacking the consensus sequence, binds to PDZ1 of SAP90 with an affinity of 160 muM. The NMR data illustrate that although all three peptides occupy the binding groove capped by the GLGF loop of PDZ1, specific differences are present, consistent with the variation in binding affinities.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *synapse-associated protein 90 SAP90 --analysis; *synapse-associated protein 90 SAP90 --characterization; *synapse-associated protein 90 SAP90 --PDZ1 domain; *fluorescence anisotropy --analytical method; *fluorescence anisotropy --Spectrum Analysis Techniques; *Bruker Avance 600 spectrometer --laboratory equipment; *Bruker Avance 600 spectrometer --Bruker; *NMR --analytical method; *NMR --Spectrum Analysis Techniques; *SPEX 1681 Fluorolog spectrofluorometer --laboratory equipment

2002

Structure-Assigned Optical Spectra of Single-Walled Carbon Nanotubes

Sergei M.Bachilo, Michael S.Strano, Carter Kittrell, Robert H.Hauge, Richard E,Smalley, R.bruce Weisman

SCIENCE,20 December 2002,Volume 298,pp.2361-2366

 

2002

Structural independence of the two EF-hand domains of caltractin.

Chazin, Walter J.; Fagan, Patricia A.; Harper, Jeffrey F.; Hu, Haitao; Huang, Bessie; Lee, Vincent; Veeraraghavan, Sudha

Journal of Biological Chemistry , VOL. 277, NO. 32, August 9, 2002, PP. 28564-28571.

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 muM) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *yeast (Fungi); *Escherichia coli (Enterobacteriaceae) --expression system; *Bacteria; *Eubacteria; *Fungi; *Microorganisms; *Nonvascular Plants; *Plants; *microtubule; *calcium ion; *caltractin --structural independence; *caltractin --EF-hand domain; *Kar1p protein; *domain isolation --isolation method; *domain isolation --Extraction; *domain isolation --Isolation; *domain isolation --Purification and Separation Techniques; *domain subcloning --cloning method; *domain subcloning --Molecular Biology Techniques and Chemical Characterization; *filtration columns --laboratory equipment; *filtration columns --Fisher Scientific; *peptide titration --biochemical method; *peptide titration --Crystallographic Techniques; *Bruker Avance 600 spectrometer --laboratory equipment; *Bruker Avance 600 spectrometer --Bruker; *Bruker AMX 500 spectrometer --laboratory equipment; *Bruker AMX 500 spectrometer --Bruker; *Bruker AMX 600 spectrometer --laboratory equipment; *Bruker AMX 600 spectrometer --Bruker; *Bruker DRX 750 spectrometer --laboratory equipment; *Bruker DRX 750 spectrometer --Bruker; *CD spectroscopy circular dichroism spectroscopy --imaging method; *CD spectroscopy circular dichroism spectroscopy --Spectrum Analysis Techniques; *Mono Q column --laboratory equipment; *Mono Q column --Amersham Biosciences; *NMR spectroscopy nuclear magnetic resonance spectroscopy --imaging method; *NMR spectroscopy nuclear magnetic resonance spectroscopy --Spectrum Analysis Techniques; *Spex Fluorolog fluorimeter --laboratory equipment; *Spex Fluorolog fluorimeter --Spex Industries; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --separation method; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --Electrophoretic Techniques; *UV spectroscopy --imaging method; *UV spectroscopy --Spectrum Analysis Techniques

2002

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation.

Weiner, Henry; Rodriguez-Zavala, Jose S.

Biochemistry, VOL. 41, NO. 26 , July 2, 2002, PP. 8229-8237.

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B+C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The Km for propionaldehyde was similar to that of the wild-type enzyme, but the Km for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Escherichia coli (Enterobacteriaceae) --strain-BL21; *Bacteria; *Eubacteria; *Microorganisms; *aldehyde dehydrogenase --structural aspects; *beta-mercaptoethanol; *dimer-tetramer --formation; *homotetramer; *hydroxyacetophenone-Sepharose column --laboratory equipment; *imidazole; *isopropylthio-beta-D-galactoside; *propionaldehyde; *protein --overall stability; *sodium chloride; *sodium nitrate; *urea --denaturation; *NADH; *Ser500; *gel filtration --separation method; *Beckman XL-1 --laboratory equipment; *Beckman XL-1 --Beckman; *Bio-Sil Sec-250 gel filtration column --laboratory equipment; *Bio-Sil Sec-250 gel filtration column --Bio-Rad Laboratories; *Chelating-Sepharose column --laboratory equipment; * Fluorolog-3 spectrofluorometer --laboratory equipment; *PCR polymerase chain reaction --in situ recombinant gene expression detection; *PCR polymerase chain reaction --sequencing techniques; *PCR polymerase chain reaction --DNA amplification; *dimer-dimer interface; *oligomerization

2002

RET and anisotropy measurements establish the proximity of the conserved Trp17 to Ile98 and Phe99 of tear lipocalin.

Glasgow, Ben J.; Abduragimov, Adil R.; Gasymov, Oktay K.; Yusifov, Taleh N.

Biochemistry, VOL. 41, NO. 28 , July 16, 2002, PP. 8837-8848.

Previous studies suggest that the conserved Trp17 on strand A of TL has a role in lipocalin stability and interacts, directly or indirectly, with Ile98 and Phe99 on strand G to influence ligand binding. Here, we determined the proximity of Trp17 to Ile98 and Phe99. Time-resolved fluorescence experiments showed resonance energy transfer between tryptophans at positions 17 and 98. In addition, an exciton effect was discovered in CD experiments resulting from interactions of the excited states of these tryptophans. Fluorescence anisotropy values of mutants containing two tryptophans (positions 99/17 and 98/17) were lower than expected in the absence of RET, confirming that these residues are proximate in tear lipocalin. The data support a model of tear lipocalin in which Trp17 and Phe99 are close together deep in the cavity and participate in an internal hydrophobic cluster. Ile98 is proximate to Trp17 but faces toward the outside of the cavity and in the model is part of an external hydrophobic patch. Comparison with beta-lactoglobulin suggests that these motifs may have an important influence on protein stability and ligand binding in other members of the lipocalin family.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *isoleucine-98; *lipocalin --analysis; *phenylalanine-99; *tryptophan-17; *circular dichroism CD --analytical method; *circular dichroism CD --Spectrum Analysis Techniques; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *Jasco 600 spectropolarimeter --laboratory equipment; *Jasco 600 spectropolarimeter --Jasco; *Jobin Yvon-SPEX Fluorolog tau-3 spectrofluorometer --laboratory equipment; *Jobin Yvon-SPEX Fluorolog tau-3 spectrofluorometer --Jobin Yvon; *resonance energy transfer RET

2002

Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexins in neuroexocytosis.

Fasshauer, Dirk; Jahn, Reinhard; Langen, Ralf; Margittai, Martin; Pabst, Stefan; Vainius, Darius

Journal of Biological Chemistry, VOL. 277, NO. 10, March 8, 2002, PP. 7838-7848

The Ca2+-triggered release of neurotransmitters is mediated by fusion of synaptic vesicles with the plasma membrane. The molecular machinery that translates the Ca2+ signal into exocytosis is only beginning to emerge. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin, SNAP-25, and synaptobrevin are central components of the fusion apparatus. Assembly of a membrane-bridging ternary SNARE complex is thought to initiate membrane merger, but the roles of other factors are less understood. Complexins are two highly conserved proteins that modulate the Ca2+ responsiveness of neurotransmitter release. In vitro, they bind in a 1:1 stoichiometry to the assembled synaptic SNARE complex, making complexins attractive candidates for controlling the exocytotic fusion apparatus. We have now performed a detailed structural, kinetic, and thermodynamic analysis of complexin binding to the SNARE complex. We found that no major conformational changes occur upon binding and that the complexin helix is aligned antiparallel to the four-helix bundle of the SNARE complex. Complexins bound rapidly (apprxeq5X107 M-1 s-1) and with high affinity (apprxeq10 nM), making it one of the fastest protein-protein interactions characterized so far in membrane trafficking. Interestingly, neither affinity nor binding kinetics was substantially altered by Ca2+ ions. No interaction of complexins was detectable either with individual SNARE proteins or with the binary syntaxincntdotSNAP-25 complex. Furthermore, complexin did not promote the formation of SNARE complex oligomers. Together, our data suggest that complexins modulate neuroexocytosis after assembly of membrane-bridging SNARE complexes.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Nervous System (Neural Coordination); *plasma membrane; *synapse --nervous system; *synaptic vesicles --nervous system; *calcium ion; *complexins --modulatory role; *neurotransmitters; *soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex SNARE complex --rapid binding; *soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex SNARE complex --selective binding; *synaptobrevin; *syntaxin; *SNAP-25 synaptosomal associated protein of 25 kiloDaltons ; *fluorescence anisotropy measurement --measurement method; *fluorescence anisotropy measurement --Spectrum Analysis Techniques; *ion exchange chromatography --liquid chromatography; *ion exchange chromatography --purification method; *size exclusion chromatography --liquid chromatography; *size exclusion chromatography --purification method; *stopped-flow measurement --measurement method; *stopped-flow measurement --Molecular Biology Techniques and Chemical Characterization; *AKTA system --laboratory equipment; *AKTA system --Amersham Biosciences Inc.; *Bruker EMX spectrometer --laboratory equipment; *Bruker EMX spectrometer --Bruker; * Fluorolog-3 --laboratory equipment; * Fluorolog-3 --Jobin Yvon Spex; *FluoroMax-2 --laboratory equipment; *FluoroMax-2 --Jobin Yvon Spex; *PD-10 column --laboratory equipment; *PD-10 column --Amersham Biosciences Inc.; *QuikChange site-directed mutagenesis kit --laboratory kit; *QuikChange site-directed mutagenesis kit --Stratagene; *SX.18MV stopped-flow instrument --laboratory equipment; *SX.18MV stopped-flow instrument --Applied Photophysics; *neuroexocytosis

2002

Quantitative determination of binding affinity of delta-subunit in Escherichia coli F1-ATPase. Effects of mutation, Mg2+, and pH on Kd.

Senior, Alan E.; Weber, Joachim; Wilke-Mounts, Susan

Journal of Biological Chemistry, VOL. 277, NO. 21, May 24, 2002, PP. 18390-18396

To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of delta-subunit to Escherichia coli F1-ATPase. The signal used is that of the natural tryptophan at residue delta28, which is enhanced by 50% upon binding of delta-subunit to alpha3beta3gammaepsilon complex. Kd for delta binding is 1.4 nM, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain. Only one site for delta binding was detected. The deltaW28L mutation increased Kd to 4.6 nM, equivalent to a loss of 2.9 kJ/mol binding energy. While this was insufficient to cause detectable functional impairment, it did facilitate preparation of delta-depleted F1. The alphaG29D mutation reduced Kd to 26 nM, equivalent to a loss of 7.2 kJ/mol binding energy. This mutation did cause serious functional impairment, referable to interruption of binding of delta to F1. Results with the two mutants illuminate how finely balanced is the stator resistance function. delta' fragment, consisting of residues delta1-134, bound with the same Kd as intact delta, showing that, at least in absence of Fo subunits, the C-terminal domain of delta contributes zero binding energy. Mg2+ ions had a strong effect on increasing delta binding affinity, supporting the possibility of bridging metal ion involvement in stator function. High pH environment greatly reduced delta binding affinity, suggesting the involvement of protonatable side-chains in the binding site.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *magnesium(II) ion; *ATP synthase --assay; *ATP synthase --delta subunit; *ATP synthase --mutation; *binding assay --analytical method; *binding assay --Bioassays/Physiological Analysis; *fluorimetry --analytical method; *fluorimetry --Bioassays/Physiological Analysis; *SPEX Fluorolog 2 spectrofluorometer --laboratory equipment

2002

Phosphorylation of eukaryotic initiation factor 4E markedly reduces its affinity for capped mRNA.

Scheper, Gert C.; van Kollenburg, Barbara; Goss, Dixie J.; Hu, Jianzhong; Luo, Yunjing; Proud, Christopher G.

Journal of Biological Chemistry, VOL. 277, NO. 5, February, 2002, PP. 3303-3309

In eukaryotes, a key step in the initiation of translation is the binding of the eukaryotic initiation factor 4E (eIF4E) to the cap structure of the mRNA. Subsequent recruitment of several components, including the small ribosomal subunit, is thought to allow migration of initiation complexes and recognition of the initiation codon. Mitogens and cytokines stimulate the phosphorylation of eIF4E at Ser209, but the functional consequences of this modification have remained a major unresolved question. Using fluorescence spectroscopy and surface plasmon resonance techniques, we show that phosphorylation of eIF4E markedly reduces its affinity for capped RNA, primarily due to an increased rate of dissociation. Variant eIF4E proteins harboring negatively charged acidic residues at position 209 also showed decreased binding to capped RNA. Furthermore, a basic residue at position 159 was shown to be essential for cap binding. Although eIF4E-binding protein 1 greatly stabilized binding of phosphorylated eIF4E to capped RNA, in the presence of eIF4E-binding protein 1 the phosphorylated form still dissociated faster compared with nonphopshorylated eIF4E. The implications of our findings for the mechanism of translation initiation are discussed.

DESCRIPTOR(S)- *Cell Biology; *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *eukaryote (Organisms); *Escherichia coli (Enterobacteriaceae) --expression system; *293 cell line (Hominidae) --human embryonic kidney cells; *293 cell line (Hominidae) --transfected; *Animals; *Bacteria; *Chordates; *Eubacteria; *Humans; *Mammals; *Microorganisms; *Primates; *Vertebrates; *eukaryotic initiation factor-4E --analysis; *eukaryotic initiation factor-4E --capped mRNA affinity; *eukaryotic initiation factor-4E --phosphorylation; *mRNA messenger RNA --analysis; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *surface plasmon resonance spectroscopy --analytical method; *surface plasmon resonance spectroscopy --Spectrum Analysis Techniques; *BIAcore 3000 system --laboratory equipment; *BIAcore 3000 system --BIAcore; *Spex Fluorolog 2 spectrofluorimeter --laboratory equipment

2002

Partial donor-donor energy migration (PDDEM) as a fluorescence spectroscopic tool for measuring distances in biomacromolecules.

Johansson, Lennart B.-A.; Kalinin, Stanislav V.; Molotkovsky, Julian G.

Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy, VOL. 58A, NO. 5, 15 March, 2002, PP. 1087-1097.

A theoretical model is presented, tested and applied for determining the rates of energy migration and distances within pairs of chemically identical fluorophores, so-called donors (D), which are exposed to different physical properties. The model is a general extension of the recently developed donor-donor energy migration (DDEM) model (J. Chem. Soc., Faraday Trans. 92 (1996) 1563; J. Chem. Phys. 105 (1996) 10896) that applies to examining structure-function of biomacromolecules, such as proteins. Most fluorescent groups of the same kind incorporated at different positions (alpha and beta) in a macromolecule exhibit shifts of the absorption and/or emission spectra, as well as different relaxation rates of the photophysics. As a consequence, the energy migration between the Dalpha and Dbeta groups will be partially reversible. We refer to this case, as the partial donor-donor energy migration (PDDEM). The models of PPDEM presented can be used for analysing time-resolved fluorescence relaxation, as well as fluorescence depolarisation experiments. To explore the limitations of the PDDEM model, we have generated and re-analysed synthetic data that mimic time-correlated single photon counting (TCSPC) experiments. It was found that slow and fast rates of energy migration are most accurately recovered from the fluorescence relaxation and the depolarisation experiments, respectively. At comparable transfer and fluorescence rates, both kinds of experiments are equally useful. Real experiments on PDDEM were performed on an asymmetrically quenched bichromophoric molecule (1,32-dihydroxy-dotriacontane-bis-(Rhodamine 101) ester), that spans across the lipid bilayer of a vesicle. The depolarisation data were analysed by the PDDEM model and provide a distance between Rhodamine 101 groups, which agrees with independent studies.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Mathematical Biology (Computational Biology); *Methods and Techniques; *fluorophore --analysis; *fluorophore --energy migration; *proteins --analysis; *Rhodamine-101 octadecyl ester --analysis; *Rhodamine-101 octadecyl ester --label; *Rhodamine-101 octadecyl ester --Molecular Probes Inc.; *1,2-dioleoyl-sn-glycero-3-phosphocholine --reagent; *1,2-dioleoyl-sn-glycero-3-phosphocholine --Avanti Polar Lipids; *1,32-dihydroxydotriacontane-bis-(Rhodamine 101) ester --analysis; *partial donor-donor energy migration --analytical method; *partial donor-donor energy migration --mathematical method; *partial donor-donor energy migration --mathematical model; *time-correlated single photon counting --analytical method; *time-correlated single photon counting --Spectrum Analysis Techniques; *time-resolved fluorescence depolarization --analytical method; *time-resolved fluorescence depolarization --Spectrum Analysis Techniques; *time-resolved fluorescence relaxation --analytical method; *time-resolved fluorescence relaxation --Spectrum Analysis Techniques; *NanoLED pulse diode --laboratory equipment; *NanoLED pulse diode --IBH; *PRA 3000 system --laboratory equipment; *SPEX Fluorolog 112 instrument --laboratory equipment; *SPEX Fluorolog 112 instrument --SPEX; *energy migration; *intramolecular distances; *lipid vesicle

2002

Multiple conformations of NAD and NADH when bound to human cytosolic and mitochondrial aldehyde dehydrogenase.

Hammen, Philip K.; Allali-Hassani, Abdellah; Hallenga, Klaas; Hurley, Thomas D.; Weiner, Henry

Biochemistry, VOL. 41, NO. 22, June 4, 2002, PP. 7156-7168

Crystallographic analysis revealed that the nicotinamide ring of NAD can bind with multiconformations to aldehyde dehydrogenase (ALDH) (Ni, L., Zhou, J., Hurley, T. D., and Weiner, H. (1999) Protein Sci. 8, 2784-2790). Electron densities can be defined for two conformations, neither of which appears to be compatible with the catalytic reaction. In one conformation, it would prevent glutamate 268 from functioning as a general base needed to activate the catalytic nucleophile, cysteine 302. In the other confromation, the nicotinamide is too far from the enzyme-substrate adduct for efficient hydride transfer. In this study, NMR and fluorescence spectroscopies were used to demonstrate that NAD and NADH bind to human liver cytosol and mitochondrial ALDH such that the nicotinamide samples a population of conformations while the adenosine region remains relatively immobile. Although the nicotinamide possesses extensive conformational heterogeneity, the catalyzed reaction leads to the stereospecific transfer of hydride to the coenzyme. Mobility allows the nicotinamide to move into position to be reduced by the enzyme-substrate adduct. Although the reduced nicotinamide ring retains mobility after NADH formation, the extent of the motion is less than that of NAD. It appears that after reduction the population of favored nicotinamide conformations shifts toward those that do not interfere with the ability of the enzyme to release the reaction product. In the case of the mitochondrial, but not the cytosolic, enzyme this change in conformational preference is promoted by the presence of Mg2+ ions. Coenzyme conformational mobility appears to be beneficial to catalysis by ALDH throughout the catalytic cycle.

DESCRIPTOR(S)- *Cell Biology; *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *cytosol; *mitochondria; *aldehyde dehydrogenase; *cysteine-302; *glutamate-268; *magnesium ion; *nicotinamide; *NAD --conformation; *NAD --Sigma; *NADH --conformation; *NADH --Sigma; *crystallographic analysis --visualization method; *crystallographic analysis --Crystallographic Techniques; *fluorescence spectroscopy --imaging method; *fluorescence spectroscopy --Spectrum Analysis Techniques; * Fluorolog-3 spectrofluorometer --laboratory equipment; * Fluorolog-3 spectrofluorometer --Horiba; *NMR spectroscopy --imaging method; *NMR spectroscopy --Spectrum Analysis Techniques; *catalysis

2002

Location and dynamics of tryptophan in transmembrane alpha-helix peptides: A fluorescence and circular dichroism study.

de Foresta, Beatrice; Gallay, Jacques; Tortech, Ludovic; Vincent, Michel

European Biophysics Journal, VOL. 31, NO. 3, June, 2002, PP. 185-197

Amphiphilic and hydrophobic peptides play a key role in many biological processes. We have developed a reference system for evaluating the insertion of such peptides bearing Trp fluorescent reporter groups into membrane mimetic systems. This system involves a set of six 25-amino acid synthetic peptides that are models of transmembrane alpha-helices. They are Lys-flanked polyLeu sequences, each containing a single Trp residue at a different position (Pi, with i=3, 5, 7, 9, 11 and 13). These peptides were inserted into micelles of a non-ionic detergent, dodecylmaltoside (DM). We analyzed this system by use of circular dichroism and steady-state and time-resolved fluorescence in combination with Trp quenching with two brominated DM analogs. We found significant variations in the Trp emission maximum according to its position in each peptide (from 327 to 313 nm). This is consistent with the radial insertion of the peptides within DM micelles. We observed characteristic patterns of fluorescence quenching of these peptides in mixed micelles of DM, with either 7,8-dibromododecylmaltoside (BrDM) or 10,11-dibromoundecanoylmaltoside (BrUM), that reflect differences in the accessibility of the Trp residue to the bromine atoms located on the detergent acyl chain. In the isotropic reference solvent, methanol, the alpha-helix content was high and identical (apprx76%) for all peptides. In DM micelles, the alpha-helix content for P9 to P13 was similar to that in methanol, but slightly lower for P3 to P7. The fluorescence intensity decays were heterogeneous and depended upon the position of the Trp. The Trp dynamics of each peptide are described by sub-nanosecond and nanosecond rotational motions that were significantly lower than those observed in methanol. These results, which precisely describe structural, dynamic and microenvironment parameters of peptide Trp in micelles according to its depth, should be useful for describing the interactions of peptides of biological interest with micelles.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *dodecylmaltoside micelles DM micelles --membrane model; *dodecylmaltoside micelles DM micelles --Calbiochem; *methanol --isotropic reference solvent; *methanol --Merck; *transmembrane alpha-helix peptides --alpha-helix content; *transmembrane alpha-helix peptides --synthetic models; *transmembrane alpha-helix peptides --Research Genetics; *tryptophan --dynamics; *tryptophan --fluorescence quenching; *tryptophan --location; *10,11-dibromoundecanoylmaltoside BrUM --brominated dodecylmaltoside analog; *7,8-dibromododecylmaltoside BrDM --brominated dodecylmaltoside analog; *circular dichroism --visualization method; *circular dichroism --Spectrum Analysis Techniques; *double monochromator UV-DH10 --laboratory equipment; *double monochromator UV-DH10 --Jobin Yvon; *single monochromator UV-H10 --laboratory equipment; *single monochromator UV-H10 --Jobin Yvon; *steady-state fluorescence measurement --visualization method; *steady-state fluorescence measurement --Spectrum Analysis Techniques; *synchrotron radiation machine Super-ACO --laboratory equipment; *time-resolved fluorescence measurement --visualization method; *time-resolved fluorescence measurement --Spectrum Analysis Techniques; *Hamamatsu MCP-PMT detector --laboratory equipment; *Hamamatsu MCP-PMT detector --model R3809U-02; *Hamamatsu MCP-PMT detector --Hamamatsu; *HP 8452A diode array spectrophotometer --laboratory equipment; *HP 8453 diode array spectrophotometer --laboratory equipment; *Jobin Yvon CD6 spectrodichrograph --laboratory equipment; *Jobin Yvon CD6 spectrodichrograph --Jobin Yvon; *Spex Fluorolog spectrofluorometer --laboratory equipment

2002

Kinetics and mechanism of long-chain fatty acid transport into phosphatidylcholine vesicles from various donor systems.

Hauser, Helmut; Baici, Antonio; Schulthess, Georg; Thomas, Richard M.; Werder, Moritz

Biochemistry, VOL. 41, NO. 5, February 5, 2002, PP. 1591-1601

The kinetics of long-chain fatty acid (FA) transfer from three different donor systems to unilamellar egg phosphatidylcholine (EPC) vesicles containing the pH-sensitive fluorophore pyranine in the vesicle cavity were determined. The transfer of long-chain FA from three FA donors, FA vesicles, unilamellar EPC vesicles containing FA, and bovine serum albumin-FA complexes to pyranine-containing EPC vesicles is a true first-order process, indicating that the FA transfer proceeds through the aqueous phase and not through collisional contacts between the donor and acceptor. A collisional mechanism would be at least bimolecular, giving rise to second-order kinetics. Evidence from stopped-flow fluorescence spectroscopy using the pyranine assay (as developed by Kamp, F., and Hamilton, J. A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370) shows that the transverse or flip-flop motion of long-chain FA (from 14 to 22 C atoms) is immeasurably fast in both small and large unilamellar EPC vesicles and characterized by half-times t1/2<5 ms. The rate-limiting step of FA transfer from these different donor systems to pyranine-containing EPC vesicles is the dissociation or desorption of the FA molecule from the donor. The desorption of the FA molecule is chain-length-dependent, confirming published data (Zhang et al. (1996) Biochemistry 35, 16055-16060): the first-order rate constant k1 decreases by a factor of about 10 with elongation of the FA chain by two CH2 groups. Similar rates of desorption are observed for the transfer of oleic acid from the three donors to pyranine-containing EPC vesicles with rate constants k1 ranging from 0.4 to 1.3 s-1. We also show that osmotically stressed, pyranine-containing EPC vesicles can give rise to artifacts. In the presence of a chemical potential gradient across the lipid bilayer of these vesicles, fast kinetic processes are observed with stopped-flow fluorescence spectroscopy which are probably due to electrostatic and/or osmotic effects.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *fatty acid donors; *fatty acids --membrane transport studies; *fatty acids --metabolism; *fatty acids --molecular analysis; *fluorophores --uses; *lipid bilayers --analysis; *lipid bilayers --chemical potential gradients; *lipids; *long-chain fatty acids --membrane transport studies; *long-chain fatty acids --metabolism; *long-chain fatty acids --molecular analysis; *phosphatidylcholine vesicles --analysis; *stopped-flow fluorescence spectroscopy --analytical method; *stopped-flow fluorescence spectroscopy --applications; *stopped-flow fluorescence spectroscopy --Spectrum Analysis Techniques; *thin layer chromatography --analytical method; *thin layer chromatography --liquid chromatography; * Fluorolog-2 double-monochromator spectrofluorimeter --laboratory equipment; * Fluorolog-2 double-monochromator spectrofluorimeter --Spex Industries Inc.; *electrostatic effects; *model systems --applications; *osmotic effects; *thermodynamics

2002

Isolation and characterization of rhodanese intermediates during thermal inactivation and their implications for the mechanism of protein aggregation.

Horowitz, Paul M.; Bhattacharyya, Anusri Mitra

Biochemistry, VOL. 41, NO. 1, January 8, 2002, PP. 422-429

The initial steps of heat-induced inactivation and aggregation of the enzyme rhodanese have been studied and found to involve the early formation of modified but catalytically active conformations. These intermediates readily form active dimers or small oligomers, as evident from there being only a small increase in light scattering and an increase in fluorescence energy homotransfer from rhodanese labeled with fluorescein. These species are probably not the domain-unfolded form, as they show activity and increased protection of hydrophobic surfaces. Cross-linking with glutaraldehyde and fractionation by gel filtration show the predominant formation of dimer during heat incubation. Comparison between the rates of aggregate formation at 50 degreeC after preincubation at 25 or 40 degreeC gives evidence of product-precursor relationships, and it shows that these dimeric or small oligomeric species are the basis of the irreversible aggregation. The thermally induced species is recognized by and binds to the chaperonin GroEL. The unfoldase activity of GroEL subsequently unfolds rhodanese to produce an inactive conformation and forms a stable, reactivable complex. The release of 80% active rhodanese upon addition of GroES and ATP indicates that the thermal incubation induces an alteration in conformation, rather than any covalent modification, which would lead to formation of irreversibly inactive species. Once oligomeric species are formed from the intermediates, GroEL cannot recognize them. Based on these observations, a model is proposed for rhodanese aggregation that can explain the paradoxical effect in which rhodanese aggregation is reduced at higher protein concentration.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *glutaraldehyde --reagent; *rhodanese --aggregation; *rhodanese --analysis; *rhodanese intermediates --characterization; *rhodanese intermediates --isolation; *ATP --analysis; *GroEL --analysis; *GroEL --chaperonin; *anisotropy measurement --measurement method; *anisotropy measurement --Spectrum Analysis Techniques; *cross-linking --biochemical method; *cross-linking --Preparatory and General Laboratory Techniques; *fluorescence spectroscopy --measurement method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *gel filtration --separation method; *gel filtration --Extraction; *gel filtration --Isolation; *gel filtration --Purification and Separation Techniques; *light scattering measurement --measurement method; *light scattering measurement --Spectrum Analysis Techniques; *rhodanese assay --enzymatic method; *rhodanese assay --Bioassays/Physiological Analysis; * Fluorolog-3 spectrofluorometer --laboratory equipment; * Fluorolog-3 spectrofluorometer --Jobin Yvon-Spex; *G-50 column --laboratory equipment; *Sephacryl S-100-SR column --laboratory equipment; *Sephacryl S-100-SR column --Sigma; *protein aggregation mechanisms; *thermal inactivation

2002

Fluorescence biosensing strategy based on energy transfer between fluorescently labeled receptors and a metallic surface.

Lopez, Gabriel P.; Buranda, Tione; Hampton, Philip D.; Perez-Luna, Victor H.; Rabinovich, Emmanuil M.; Sklar, Larry A.; Yang, Saipeng

Biosensors & Bioelectronics, VOL. 17, NO. 1-2, January, 2002, PP. 71-78

A new fluorescence-based biosensor is presented. The biosensing scheme is based on the fact that a fluorophore in close proximity to a metal film (<100 ANG) experiences strong quenching of fluorescence and a dramatic reduction in the lifetime of the excited state. By immobilizing the analyte of interest (or a structural analog of the analyte) to a metal surface and exposing it to a labeled receptor (e.g. antibody), the fluorescence of the labeled receptor becomes quenched upon binding because of the close proximity to the metal. Upon exposure to free analyte, the labeled receptor dissociates from the surface and diffuses into the bulk of the solution. This increases its separation from the metal and an increase of fluorescence intensity and/or lifetime of the excited state is observed that indicates the presence of the soluble analyte. By enclosing this system within a small volume with a semipermeable membrane, a reversible device is obtained. We demonstrate this scheme using a biotinylated self-assembled monolayer (SAM) on gold as our surface immobilized analyte analog, fluorescently labeled anti-biotin as a receptor, and a solution of biotin in PBS as a model analyte. This scheme could easily be extended to transduce a wide variety of protein-ligand interactions and other biorecognition phenomena (e.g. DNA hybridization) that result in changes in the architecture of surface immobilized biomolecules such that a change in the separation distance between fluorophores and the metal film is obtained.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Equipment, Apparatus, Devices and Instrumentation; *antibodies; *biomolecules --analysis; *biomolecules --characterization; *biomolecules --detection; *biomolecules --labeling; *fluorescent dyes --uses; *fluorophores --uses; *biosensors --analytical method; *biosensors --applications; *biosensors --blotting/hybridization/molecular probe techniques; *biosensors --construction; *biosensors --description; *biosensors --development; *biosensors --device; *biosensors --equipment; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *fluorescence-based biosensor --analytical method; *fluorescence-based biosensor --applications; *fluorescence-based biosensor --blotting/hybridization/molecular probe techniques; *fluorescence-based biosensor --construction; *fluorescence-based biosensor --description; *fluorescence-based biosensor --development; *fluorescence-based biosensor --device; *fluorescence-based biosensor --equipment; *fluorescence-based biosensor --new; *immunoassays --analytical method; *immunoassays --applications; *immunoassays --labeling; *metallic surfaces --equipment; *surface plasmon resonance --analytical method; *surface plasmon resonance --Molecular Biology Techniques and Chemical Characterization; *SPEX Fluorolog 3 instrument --laboratory equipment; *SPEX Fluorolog 3 instrument --uses; *SPEX Fluorolog 3 instrument --Instruments S. A.; *bioelectronics; *biorecognition phenomena; *biosensing schemes/strategies --applications; *biosensing schemes/strategies --descriptions; *biotechnology; *electrochemistry --applications; *energy transfer --analysis; *metals --applications; *metals --films

2002

Evidence for nonhydrogen bonded compound II in cyclic reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide.

Lopez-Garriga, Juan; Cerda, Jose; De Jesus-Bonilla, Walleska; Ramirez-Melendez, Eunice

Biopolymers, VOL. 67, NO. 3, 2002, PP. 178-185

Studies that elucidate the behavior of the hemoglobins (Hbs) and myoglobins upon reaction with hydrogen peroxide are essential to the development of oxygen carrier substitutes. Stopped-flow kinetics and resonance Raman data show that the reaction between hydrogen peroxide and oxygenated and deoxygenated ferric Hb I (oxy-and deoxy-HbI) from Lucina pectinata produce compound I and compound II ferryl species. The rate constants ratio (k23/k41) between the formation of compound II from compound I (k23) and the oxidation of the ferrous HbI (k41, i.e., 25 M-1 s-1) of 12 X 10-4 M suggests that HbI has a peroxidative capacity for removing H2O2 from solution. Resonance Raman presents the formation of both, met-aquo-HbI and compound II ferryl species in the cyclic reaction of HbI with H2O2. The ferric HbI species is maintained by the presence of H2O2; it can produce HbI compound I, or it can be reduced to a deoxy-HbI derivative to form HbI compound II upon reaction with H2O2. The compound II ferryl vibration frequency appears at 805 and 769 cm-1 for HbIFeIVdbd16O and HbIFeIVdbd18O species, respectively. This ferryl mode indicates the absence of hydrogen bonding between the carbonyl group of the distal Q64 and the HbIFeIVdbdO ferryl moiety. The observation suggests that both the trans-ligand effect and the polarizability of the HbI heme pocket are responsible for the observed ferryl oxo vibrational energy. The vibrational mode also suggests that the carbonyl group of the distal Q64 is oriented toward the iron of the heme group, increasing the distal pocket electron density.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Metabolism; *Methods and Techniques; *Lucina pectinata (Pelecypoda); *Animals; *Invertebrates; *Mollusks; *ferryl species; *heme; *hemoglobin I --ferric; *hemoglobin I --ferrous; *hydrogen peroxide --Fisher Co.; *myoglobin; *nonhydrogen bonded compound I; *nonhydrogen bonded compound II; *oxygen; *cooled CCD detector --laboratory equipment; *cooled CCD detector --Spex; *krypton laser --laboratory equipment; *resonance Raman spectroscopy --imaging method; *resonance Raman spectroscopy --Spectrum Analysis Techniques; *spectrometer --laboratory equipment; *spectrometer --Spex; *stopped-flow kinetic analysis --evaluation method; *stopped-flow kinetic analysis --Spectrum Analysis Techniques; * Fluorolog-2 scanning spectrofluorometer system --laboratory equipment; * Fluorolog-2 scanning spectrofluorometer system --Inc.; * Fluorolog-2 scanning spectrofluorometer system --On-Line Instrument Systems; *cyclic reaction

2002

Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A.

Ackerman, Eric J.; van Houten, Ben; Iakoucheva, Lilia M.; Walker, Randall K.

Biochemistry, VOL. 41, NO. 1, January 8, 2002, PP. 131-143

Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (Kd) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant kon of 9.03 X 108 M-1 s-1 and koff of 26.1 s-1. From the ratio of off-rate to on-rate, a calculated Kd of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorometry experiments was up to 3 orders of magnitude higher than previously reported values using different substrates, conditions, and assays (gel-shifts (EMSA), filter-binding, anisotrophy, and surface plasmon resonance). The same substrate DNA containing a 4-bp mismatch in the middle yielded a Kd five times higher (158 nM), indicating weaker binding by xXPA. The differences in Kd values for these two substrates were mainly attributable to the kon, rather than koff rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate Kd. Although recombinant human RPA binding to the ds 50-mer was very weak (Kd > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded Kd values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the Kd.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *human (Hominidae); *Xenopus (Salientia); *Amphibians; *Animals; *Chordates; *Humans; *Mammals; *Nonhuman Vertebrates; *Primates; *Vertebrates; *fluorescein --label; *human replication protein A hRPA --DNA repair protein; *oligonucleotides --double-stranded; *oligonucleotides --fluorescently-labeled; *oligonucleotides --single-stranded; *oligonucleotides --substrate; *oligonucleotides --Genosys Biotechnologies; *DNA --analysis; *Xenopus xeroderma pigmentosum group A protein xXPA --analysis; *Xenopus xeroderma pigmentosum group A protein xXPA --DNA repair protein; *anisotropy --analytical method; *anisotropy --Spectrum Analysis Techniques; *electrophoretic mobility shift assay EMSA --analytical method; *electrophoretic mobility shift assay EMSA --restriction fragment mapping; *filter-binding assay --bioassay method; *filter-binding assay --Bioassays/Physiological Analysis; *gel mobility shift assay --bioassay method; *gel mobility shift assay --Bioassays/Physiological Analysis; *spectrofluorometry --measurement method; *spectrofluorometry --Spectrum Analysis Techniques; *surface plasmon resonance --analytical method; *surface plasmon resonance --Spectrum Analysis Techniques; *AVIV ATF105 spectrofluorometer --laboratory equipment; *AVIV ATF105 spectrofluorometer --Aviv Instruments Inc.; *MOS-250 spectrofluorometer --laboratory equipment; *MOS-250 spectrofluorometer --Molecular Kinetics Inc.; *SPEX Fluorolog II spectrofluorometer --laboratory equipment; *dissociation constant; *equilibrium studies; *nucleotide excision repair NER ; *stop-flow kinetic studies

2002

Enzyme assays by fluorescence polarization in the presence of polyarginine: Study of kinase, phosphatase, and protease reactions.

Nikiforov, Theo T.; Bi, Xiahui; Simeonov, Anton

Analytical Biochemistry, VOL. 304, NO. 2, May 15, 2002, PP. 193-199

We have previously reported that the kinase catalyzed conversion of fluorescently labeled phosphate acceptor peptides to the corresponding phosphopeptides can be conveniently followed by measuring the fluorescence polarization signal in the presence of polyarginine. In the present work, we demonstrate that the method can be used for other enzymes besides kinases, such as phosphatases and proteases. By adjustment of the ionic strength of the buffer it is possible to use this method in cases where both the substrate and the enzymatic product are highly negatively charged. All of these enzymatic transformations can be followed in real time, by performing the reactions in the presence of polyarginine and continuously measuring the fluorescence polarization signal. Polyarginine was found to have no effect on the rate of enzymatic conversion of the protease studied (cathepsin G), but its presence decreased the observed rate of phosphorylation by protein kinase A, presumably by decreasing the concentration of free ATP in the reaction solution. Leukocyte antigen related phosphatase catalyzed dephosphorylation reactions were faster in the presence of polyarginine. For all three enzymes, the reaction rates in the presence of polyarginine were found to be sensitive to the presence of known enzyme inhibitors, but the IC50 values of the kinase inhibitors H-89 and PKI were higher in the presence than in the absence of polyarginine.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *polyarginine; *fluorescence polarization --enzyme assay; *fluorescence polarization --fluorometry; * Fluorolog-3 fluorescence spectrometer --JY Horiba; * Fluorolog-3 fluorescence spectrometer --Spectrum Analysis Techniques; *Fluoromax-2 fluorescence spectrometer --JY Horiba; *Fluoromax-2 fluorescence spectrometer --Spectrum Analysis Techniques; *analytical biochemistry; *kinase reactions; *phosphatase reactions; *protease reaction

2002

Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor.

Miller, Laurence J.; Harikumar, Kaleeckal G.; Pinon, Delia I.; Prendergast, Franklyn G.; Wessels, William S.

Journal of Biological Chemistry, VOL. 277, NO. 21, May 24, 2002, PP. 18552-18560

Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa488, nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodile and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *acrylodan --fluorophore; *cholecystokinin receptor; *nitrobenzoxadiazolyl --fluorophore; *peptide agonists; *peptide antagonists; *Alexa-468 --fluorophore; *fluorescence spectra --analytical method; *fluorescence spectra --Spectrum Analysis Techniques; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *SPEX Fluorolog spectrofluorophotometer --laboratory equipment; *SPEX Fluorolog spectrofluorophotometer --SPEX

2002

Dynamics of energy transfer from lycopene to bacteriochlorophyll in genetically-modified LH2 complexes of Rhodobacter sphaeroides.

Sundstrom, V.; Billsten, H. Horvin; Garcia-Asua, G.; Hashoj, L.; Herek, J. L.; Hunter, C. N.; Polivka, T.

Biochemistry, VOL. 41, NO. 12, March 26, 2002, PP. 4127-4136

LH2 complexes from Rb. sphaeroides were modified genetically so that lycopene, with 11 saturated double bonds, replaced the native carotenoids which contain 10 saturated double bonds. Tuning the S1 level of the carotenoid in LH2 in this way affected the dynamics of energy transfer within LH2, which were investigated using both steady-state and time-resolved techniques. The S1 energy of lycopene in n-hexane was determined to be apprx12 500+-150 cm-1, by direct measurement of the S1-S2 transient absorption spectrum using a femtosecond IR-probing technique, thus placing an upper limit on the S1 energy of lycopene in the LH2 complex. Fluorescence emission and excitation spectra demonstrated that energy can be transferred from lycopene to the bacteriochlorophyll molecules within this LH2 complex. The energy-transfer dynamics within the mutant complex were compared to wild-type LH2 from Rb. sphaeroides containing the carotenoid spheroidene and from Rs. molischianum, in which lycopene is the native carotenoid. The results show that the overall efficiency for CrtfwdarwB850 energy transfer is apprx80% in lyco-LH2 and apprx95% in WT-LH2 of Rb. sphaeroides. The difference in overall CrtfwdarwBChl transfer efficiency of lyco-LH2 and WT-LH2 mainly relates to the low efficiency of the Crt S1fwdarwBChl pathway for complexes containing lycopene, which was 20% in lyco-LH2. These results show that in an LH2 complex where the Crt S1 energy is sufficiently high to provide efficient spectral overlap with both B800 and B850 Qy states, energy transfer via the Crt S1 state occurs to both pigments. However, the introduction of lycopene into the Rb. sphaeroides LH2 complex lowers the S1 level of the carotenoid sufficiently to prevent efficient transfer of energy to the B800 Qy state, leaving only the Crt S1fwdarwB850 channel, strongly suggesting that Crt S1fwdarwBChl energy transfer is controlled by the relative Crt S1 and BChl Qy energies.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Rhodobacter molischianum (Purple Nonsulfur Bacteria); *Rhodobacter sphaeroides (Purple Nonsulfur Bacteria); *Bacteria; *Eubacteria; *Microorganisms; *bacteriochlorophyll; *lycopene --analysis; *LH2 complex; *absorption spectra --analytical method; *absorption spectra --Spectrum Analysis Techniques; *excitation spectra --analytical method; *excitation spectra --Spectrum Analysis Techniques; *fluorescence emission spectra --analytical method; *fluorescence emission spectra --Spectrum Analysis Techniques; *Beckman DU640 spectrophotometer --laboratory equipment; *Beckman DU640 spectrophotometer --Beckman; *Spex FluoroLOG spectrofluorometer --laboratory equipment

2002

Characterization of the unfolding of ribonuclease A by a pulsed hydrogen exchange study: Evidence for competing pathways for unfolding.

Udgaonkar, Jayant B.; Juneja, Juhi

Biochemistry, VOL. 41, NO. 8, February 26, 2002, PP. 2641-2654

The unfolding of ribonuclease A was studied in 5.2 M guanidine hydrochloride at pH 8 and 10degreeC using multiple optical probes, native-state hydrogen exchange (HX), and pulse labeling by hydrogen exchange. First, native-state HX studies were used to demonstrate that the protein exists in two slowly interconverting forms under equilibrium native conditions: a predominant exchange-incompetent N form and an alternative ensemble of conformations, NI*, in which some amide hydrogens are fully exposed to exchange. Pulsed HX studies indicated that, during unfolding, the rates of exposure to exchange with solvent protons were similar for all backbone NH probe protons. It is shown that two parallel routes of unfolding are available to the predominant N conformation as soon as it encounters strong unfolding conditions. A fraction of molecules appears to rapidly form NI* on one route. On the other route an exchange-incompetent intermediate state ensemble, IU2, is formed. The kinetics of unfolding measured by far-UV circular dichroism (CD) were faster than those measured by near-UV CD and intrinsic tyrosine fluorescence of the protein. The logarithms of the rate constants of the unfolding reaction measured by all three optical probes also showed a nonlinear dependence on GdnHCl concentration. All of the data suggest that NI* and IU2 are nativelike in their secondary and tertiary structures. While NI* unfolds directly to the fully exchange-competent unfolded state (U), IU2 forms another intermediate IU3 which then unfolds to U. IU3 is devoid of all native alpha-helical secondary structure and has only 30% of the tertiary interactions still intact. Since the rates of global unfolding measured by near-UV CD and fluorescence agree well with the rates of exposure determined for all of the backbone NH probe protons, it appears that the rate-limiting step for the unfolding of RNase A is the dissolution of the entire native tertiary structure and penetration of water into the hydrophobic core.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *guanidine hydrochloride --reagent; *ribonuclease A RNase A --type XIIA; *ribonuclease A RNase A --unfolding characterization; *ribonuclease A RNase A --Sigma Chemical Co.; *ribonuclease A unfolding intermediates --secondary structure; *ribonuclease A unfolding intermediates --tertiary structure; *far-UV circular dichroism far-UV CD --measurement method; *far-UV circular dichroism far-UV CD --optical probe; *far-UV circular dichroism far-UV CD --Spectrum Analysis Techniques; *intrinsic tyrosine fluorescence analysis --measurement method; *intrinsic tyrosine fluorescence analysis --optical probe; *intrinsic tyrosine fluorescence analysis --Spectrum Analysis Techniques; *native-state hydrogen exchange analysis native-state HX analysis --biochemical method; *native-state hydrogen exchange analysis native-state HX analysis --Molecular Biology Techniques and Chemical Characterization; *near-UV circular dichroism near-UV CD --measurement method; *near-UV circular dichroism near-UV CD --optical probe; *near-UV circular dichroism near-UV CD --Spectrum Analysis Techniques; *pulsed hydrogen exchange analysis pulsed HX analysis --biochemical method; *pulsed hydrogen exchange analysis pulsed HX analysis --Molecular Biology Techniques and Chemical Characterization; *Amicon YM 3 ultrafiltration membrane --laboratory equipment; *Amicon YM 3 ultrafiltration membrane --Amicon; *Centricon 10 microconcentration tubes --laboratory equipment; *DynaPro-99 machine --laboratory equipment; *DynaPro-99 machine --Protein Solutions Ltd.; *Jasco J720 spectropolarimeter --laboratory equipment; *Jasco J720 spectropolarimeter --Jasco; *NMR --detection method; *NMR --Imaging Techniques; *NMR --Spectrum Analysis Techniques; *Spex DM3000 Fluorolog spectrofluorimeter --laboratory equipment; *Varian 600 MHz NMR spectrometer --laboratory equipment; *Varian 600 MHz NMR spectrometer --Varian; *competing unfolding pathways; *unfolding kinetics

2002

Bioluminescence resonance energy transfer assays for protein-protein interactions in living cells.

Xu, Yao; Johnson, Carl Hirschie; Piston, David

Methods in Molecular Biology, VOL. 183, 2002, PP. 121-133.

Biochemistry and Molecular Biophysics; *Equipment, Apparatus, Devices and Instrumentation; *Methods and Techniques; *Aequorea victoria (Cnidaria); *Escherichia coli (Enterobacteriaceae) --strain-BL21; *Synechococcus sp. (Chroococcales) --strain-PCC 7942; *Animals; *Bacteria; *Cyanobacteria; *Eubacteria; *Invertebrates; *Microorganisms; *bioluminescence resonance energy transfer assay --bioassay techniques; *bioluminescence resonance energy transfer assay --laboratory techniques; *interference filters --laboratory equipment; *FB12 luminometer --laboratory equipment; *FB12 luminometer --Zylux; *SPEX fluorolog spectrofluorometer --laboratory equipment; *protein-protein interactions; *Book Chapter

2002

Aromatic compounds in molecular phase of Baltic amber: Synchronous luminescence analysis.

Matuszewska, Aniela; Czaja, Maria

Talanta , VOL. 56, NO. 6, 8 April, 2002, PP. 1049-1059

Synchronous luminescence analysis was performed in order to identify aromatic compounds in solvent extracts of Baltic amber. The investigated extracts were obtained, for comparisons, as products of extraction by various techniques and solvents. Methylene chloride and ethanol were applied independently for extraction at the ambient temperature (conservative extraction), as well as at the temperature of solvent boiling (extraction in Soxhlet apparatus). Ethanol, as the solvent, was also used for extraction in an ultrasonic bath and for the decoction process. The extraction, by techniques mentioned, of the analysed amber has resulted in products generally containing the same groups of aromatics: mainly naphthalenes, phenanthrenes and anthracenes. Among phenanthrenes, in all samples the retene was also identified, being one of the characteristic links of the diagenetic chain of chemical transformations of vegetal precursors. The identification of a series of individual compounds made, using the synchronous luminescence technique, was verified by the record of conventional emission and excitation spectra. Presented identified compounds were also confirmed by the results of GC-MS analysis. The luminescence analysis was also performed comparatively for fossil resin from Galicia, Spain (Cretaceous) older than Baltic amber (Tertiary, Eocene). The obtained preliminary results of synchronous luminescence analysis suggest the possibility of diversification in this manner of fossil resins of various ages by characterisation of aromatisation degree and alkyl substitution of aromatic rings. It is since well known that aromatisation progress is an indicator of a natural process of maturation of fossil organic matter. However, a greater number of samples should be taken to further testify to the investigations.

DESCRIPTOR(S)- *Chemistry; *Methods and Techniques; *Paleobiology; *Baltic amber; *Baltic amber molecular phase aromatic compounds --synchronous luminescence analysis; *Baltic amber solvent extracts; * Fluorolog 3-12 luminescence spectra --Molecular Biology Techniques and Chemical Characterization; * Fluorolog 3-12 luminescence spectra --Spex; *Hewlett-Packard GC-MS 5971 series apparatus --Chromatographic Techniques; *Hewlett-Packard GC-MS 5971 series apparatus --Hewlett-Packard; *Hewlett-Packard GC-MS 5971 series apparatus --Spectrum Analysis Techniques; *UV-Vis luminescence spectra --analytical method; *UV-Vis luminescence spectra --detection method; *analytical chemistry; *geochemistry

2002

A new and simple procedure for the evaluation of the association of surfactants to proteins.

Lissi, Eduardo; Abuin, Elsa; Alvarez, Carlos; Lanio, Maria E.

Journal of Biochemical and Biophysical Methods, VOL. 50, NO. 2-3, 4 January, 2002, PP. 261-268

A new and simple method useful for the evaluation of the association of surfactants to proteins is proposed. The method is based on an analysis of the effect promoted by surfactant addition upon the fluorescence intensity of the intrinsic tryptophan chromophore and its dependence with protein concentration. The proposed methodology is applied to quantify the binding of an anionic (sodium dodecylsulfate), a zwitterionic (N-hexadecyl-N,N'-dimethyl-3-ammonio-1-propane-sulfonate) and a neutral (Triton X-100, reduced) surfactant to bovine serum albumin (BSA).

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *bovine serum albumin; *proteins --molecular analysis; *surfactants --molecular analysis; *tryptophan chromophores; *fluorescence spectrometry --analytical method; *fluorescence spectrometry --applications; *fluorescence spectrometry --fluorophotometry; *Spex Fluorolog 1681 fluorescence spectrometer --laboratory equipment; *molecular associations --analysis; *molecular associations --implications; *pH

2002

A hierarchic approach to the design of hexameric helical barrels.

DeGrado, William F.; Eisenberg, David; Ghirlanda, Giovanna; Lear, James D.; Ogihara, Nancy L.

Journal of Molecular Biology, VOL. 319, NO. 1, 24 May, 2002, PP. 243-253.

The design of large macromolecular assemblies is an endeavor with implications for protein engineering as well as nanotechnology. A hierarchic approach was used to design an antiparallel hexameric, tubular assembly of helices. In previous studies, a domain-swapped, dimeric three-helix bundle was designed from first principles. In the crystal lattice, three dimers associate around a 3-fold rotational axis to form a hexameric assembly. Although this hexameric assembly was not observed in solution, it was possible to stabilize its formation by changing three polar residues per monomer to hydrophobic (two Phe and one Trp) residues. Molecular models based on the crystallographic coordinates of DSD (PDB accession code 1G6U) show that these side-chains pack in the central cavity (the "supercore") of the hexameric bundle. Analytical ultracentrifugation, fluorescence spectroscopy, CD spectroscopy, and guanidine-HCl denaturation were used to determine the assembly of the hexamer. To probe the requirements for stabilizing the hexamer, we systematically varied the polarity and steric bulk of one of the Phe residues in the supercore of the hexamer. Depending on the nature of this side-chain, it is possible to modulate the stability of the hexamer in a predictable manner. This family of hexameric proteins may provide a useful framework for the construction of proteins that change their oligomeric states in response to binding of small molecules.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *guanidine-hydrochloric acid --denaturation; *hexameric helical barrels --design; *N-hydroxybenzotriazole HObt --NovaBiochem; *2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate --NovaBiochem; *5 4-(aminomethyl)-3,5-bis(methoxy)-phenoxy valeric acid resin --NovaBiochem; *9-fluorenyl-methyoxycarbonyl --NovaBiochem; *analytical ultracentrifugation --analytical method; *analytical ultracentrifugation --centrifugation; *dual-syringe automated titrator --laboratory equipment; *dual-syringe automated titrator --Aviv Associates; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *nanotechnology --analytical method; *protein engineering --genetic engineering; *protein engineering --molecular method; *Aviv Associates fluorometer --laboratory equipment; *Aviv Associates fluorometer --Aviv Associates; *ABI 433 synthesizer --laboratory equipment; *ABI 433 synthesizer --PE Applied Biosystems; *Beckman XLI analytical centrifuge --laboratory equipment; *Beckman XLI analytical centrifuge --Beckman; *C-18 column --Vydac; *CD spectroscopy --spectroscopic method; *CD spectroscopy --Spectrum Analysis Techniques; *Discover --computer software; *Discover --Accylrys; * Fluorolog spectrofluorometer --laboratory equipment; * Fluorolog spectrofluorometer --Fluorolog; *FPLC system --laboratory equipment; *FPLC system --Amersham Pharmacia Biosystems; *Igor Pro --computer software; *Igor Pro --Inc.; *Igor Pro --WaveMetrics; *Peltier thermostated cell holder --laboratory equipment; *Peltier thermostated cell holder --Peltier; *Superdex 75 column --laboratory equipment; *oligomeric states; *tubular assembly

2002

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli.

Daunert, Sylvia; Ensor, C. Mark; Goldsmith, Elizabeth S.; Salins, Lyndon L. E.

Analytical and Bioanalytical Chemistry, VOL. 372, NO. 1, January, 2002, PP. 174-180.

A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible. The versatility of NBP was demonstrated by its use in three different assay formats. When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel. The NBP gene was overexpressed in E. coli and the protein purified in a single step using perfusion anion-exchange chromatography. A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide (MDCC). In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel. A response curve for nickel using NBP-MDCC revealed a detection limit of 8X10-8 mol L-1. NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats. Detection limits for nickel using these formats were also in the submicromolar range. Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel. The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel. These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel.

DESCRIPTOR(S)- *Equipment, Apparatus, Devices and Instrumentation; *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Pollution Assessment Control and Management; *Escherichia coli (Enterobacteriaceae) --strain-B834; *Bacteria; *Eubacteria; *Microorganisms; *cadmium; *cobalt; *copper; *cysteine; *iron; *manganese; *nickel --detection; *nickel --pollutant; *nickel --toxin; *nickel binding protein NBP --fluorescently labeled; *nickel binding protein-N- 2-(1-maleimidyl)ethyl -7-(diethylamino)coumarin-3-carboxamide NBP-MDCC --analysis; *N- 2-(1-maleimidyl)ethyl -7-(diethylamino)coumarin-3-carboxamide MDCC --fluorophore; *environmental monitoring --monitoring method; *environmental monitoring --Preparatory and General Laboratory Techniques; *fiber optic bundle --laboratory equipment; *fiber optic bundle --Oriel; *fluorescence-based sensing system --laboratory equipment; *perfusion anion-exchange chromatography --purification method; *perfusion anion-exchange chromatography --Chromatographic Techniques; *spectrofluorometric assay --analytical method; *spectrofluorometric assay --Spectrum Analysis Techniques; *BioCAD Sprint Perfusion Chromatography System --laboratory equipment; *BioCAD Sprint Perfusion Chromatography System --PerSeptive Biosystems; *Cytofluor series 4000 fluorescence multi-well plate reader --laboratory equipment; *Cytofluor series 4000 fluorescence multi-well plate reader --Applied Biosystems; *Fluoronunc Maxisorp 96-well white microtiter plates --laboratory equipment; *Fluoronunc Maxisorp 96-well white microtiter plates --Fisher Scientific; *Hewlett-Packard model 8453 diode array spectrophotometer --laboratory equipment; *Hewlett-Packard model 8453 diode array spectrophotometer --Hewlett-Packard; *Spex Industries Fluorolog-2 spectrofluorimeter --laboratory equipment; *Spex Industries Fluorolog-2 spectrofluorimeter --Spex Industries; *detection limits; *fluorescence signal quenching