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

2001

The neurokinin A receptor activates calcium and cAMP responses through distinct conformational states.

Galzi, Jean-Luc; Bucher, Bernard; Edelstein, Stuart J.; Ilien, Brigitte; Palanche, Tania; Reck, Marie-Pierre; Zoffmann, Sannah

Journal of Biological Chemistry, VOL. 276, NO. 37, September 14, 2001, PP. 34853-34861

G protein-coupled receptors are thought to mediate agonist-evoked signal transduction by interconverting between discrete conformational states endowed with different pharmacological and functional properties. In order to address the question of multiple receptor states, we monitored rapid kinetics of fluorescent neurokinin A (NKA) binding to tachykinin NK2 receptors, in parallel with intracellular calcium, using rapid mixing equipment connected to real time fluorescence detection. Cyclic AMP accumulation responses were also monitored. The naturally truncated version of neurokinin A (NKA-(4-10)) binds to the receptor with a single rapid phase and evokes only calcium responses. In contrast, full-length NKA binding exhibits both a rapid phase that correlates with calcium responses and a slow phase that correlates with cAMP accumulation. Furthermore, activators (phorbol esters and forskolin) and inhibitors (Ro 31-8220 and H89) of protein kinase C or A, respectively, exhibit differential effects on NKA binding and associated responses; activated protein kinase C facilitates a switch between calcium and cAMP responses, whereas activation of protein kinase A diminishes cAMP responses. NK2 receptors thus adopt multiple activatable, active, and desensitized conformations with low, intermediate, or high affinities and with distinct signaling specificities.

DESCRIPTOR(S)- *Cell Biology; *Enzymology (Biochemistry and Molecular Biophysics); *Equipment, Apparatus, Devices and Instrumentation; *Methods and Techniques; *HEK 293 cell line (Hominidae) --human embryonic kidney cells; *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *calcium; *cAMP cyclic AMP --accumulation; *forskolin --enzyme activator; *forskolin --Calbiochem; *forskolin --Sigma; *neurokinin A receptor --calcium activator; *phorbol esters --enzyme activator; *phorbol esters --Calbiochem; *phorbol esters --Sigma; *protein kinase A; *protein kinase C; *tachykinin NK2 receptors tachykinin neurokinin A receptors ; *G protein-coupled receptors; *H89 --enzyme inhibitor-drug; *H89 --Calbiochem; *H89 --Sigma; *Ro 31-8220 --enzyme inhibitor-drug; *Ro 31-8220 --Calbiochem; *Ro 31-8220 --Sigma; *real time fluorescence detection --detection method; *real time fluorescence detection --Spectrum Analysis Techniques; *Applied Photophysics SK1E rapid mixing equipment --laboratory equipment; *Applied Photophysics SK1E rapid mixing equipment --Applied Photophysics; *SPEX fluorolog 2 spectrofluorometer --laboratory equipment; *conformational states; *signal transduction

2001

Solvation energetics and conformational change in EF-hand proteins.

Desjarlais, John R.; Ababou, Abdessamad

Protein Science, VOL. 10, NO. 2, February, 2001, PP. 301-312

Calmodulin and other members of the EF-hand protein family are known to undergo major changes in conformation upon binding Ca2+. However, some EF-hand proteins, such as calbindin D9k, bind Ca2+ without a significant change in conformation. Here, we show the importance of a precise balance of solvation energetics to conformational change, using mutational analysis of partially buried polar groups in the N-terminal domain of calmodulin (N-cam). Several variants were characterized using fluorescence, circular dichroism, and NMR spectroscopy. Strikingly, the replacement of polar side chains glutamine and lysine at positions 41 and 75 with nonpolar side chains leads to dramatic enhancement of the stability of the Ca2+-free state, a corresponding decrease in Ca2+-binding affinity, and an apparent loss of ability to change conformation to the open form. The results suggest a paradigm for conformational change in which energetic strain is accumulated in one state in order to modulate the energetics of change to the alternative state.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *calbindin D9k; *calcium(II) ion; *calmodulin --amino-terminal domain; *calmodulin --conformational change; *calmodulin --partially buried polar groups; *glutamine; *lysine; *EF-hand protein family --conformational change; *EF-hand proteins --conformational change; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *mutational analysis --analytical method; *mutational analysis --Molecular Biology Techniques and Chemical Characterization; *AVIV 62DS CD spectrophotometer --laboratory equipment; *AVIV 62DS CD spectrophotometer --AVIV; *BioSpec-1601 --laboratory equipment; *BioSpec-1601 --SHIMADZU Scientific Instruments; * Fluorolog-3 spectrofluorimeter --laboratory equipment; *NMR spectroscopy --analytical method; *NMR spectroscopy --spectroscopic techniques: CB; *energetic strain; *solvation energetics

2001

Screening potential of solid-phase extraction and solid surface room temperature fluorimetry for polycyclic aromatic hydrocarbons in water samples.

Campiglia, Andres D.; Whitcomb, Jennifer L.

Talanta, VOL. 55, NO. 3, 13 September, 2001, PP. 509-518

The analytical potential of solid-phase extraction and room temperature fluorimetry for screening polycylic aromatic hydrocarbons in water samples is evaluated. Solid-phase extraction was performed via a syringe procedure previously reported (Talanta 52 (2000) 727). The simplicity of fluorescence measurements on the solid substrate is equivalent to solution measurements. Since oxygen quenching of fluorescence is not significant, placing the extraction membrane in the substrate holder of the spectrometer rapidly performs fluorescence measurements. Limits of detection at the pg ml-1 level were estimated for several pollutants. With a commercial spectrofluorimeter, benzo(a)pyrene was quantitatively determined at the 5 pg ml-1 concentration level. This concentration compared favorably to limits of detection estimated by laser-induced fluorimetry. A unique advantage of this approach was the possibility of adjusting the volume of extracted water to reach concentration levels below instrumental detection levels. Since SPE procedure was rapid and simple the trade-off of including an additional experimental step to lower limits of detection was advantageous. Although the selectivity of this approach was not fully investigated, our studies showed that selective excitation was sufficient to identify benzo(a)pyrene in a seven-component mixture and spiked Red River water of unknown composition.

DESCRIPTOR(S)- *Methods and Techniques; *Pollution Assessment Control and Management; *Toxicology; *anthracene --analysis; *anthracene --pollutant; *anthracene --screening; *anthracene --Aldrich; *benzo(a)pyrene --analysis; *benzo(a)pyrene --pollutant; *benzo(a)pyrene --screening; *benzo(a)pyrene --Aldrich; *chrysene --analysis; *chrysene --pollutant; *chrysene --screening; *chrysene --Aldrich; *dibenz(a,h)anthracene --analysis; *dibenz(a,h)anthracene --pollutant; *dibenz(a,h)anthracene --screening; *dibenz(a,h)anthracene --Aldrich; *indeno(1,2,3-cd)pyrene --analysis; *indeno(1,2,3-cd)pyrene --pollutant; *indeno(1,2,3-cd)pyrene --screening; *indeno(1,2,3-cd)pyrene --Aldrich; *phenanthrene --analysis; *phenanthrene --pollutant; *phenanthrene --screening; *phenanthrene --Aldrich; *polycyclic aromatic hydrocarbons --analysis; *polycyclic aromatic hydrocarbons --pollutant; *polycyclic aromatic hydrocarbons --screening; *polycyclic aromatic hydrocarbons --Aldrich; *pyrene --analysis; *pyrene --pollutant; *pyrene --screening; *pyrene --Aldrich; *solid surface room temperature fluorimetry --analytical method; *solid surface room temperature fluorimetry --fluorometry; *solid-phase extraction --extraction method; *solid-phase extraction --Extraction; *solid-phase extraction --Isolation; *solid-phase extraction --Purification and Separation Techniques; * Fluorolog-3 model FL3-11 spectrofluorimeter --laboratory equipment; * Fluorolog-3 model FL3-11 spectrofluorimeter --Jobin Yvon-Spex; *river water

2001

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac.

Laprade, Raynald; Bes, Martine; Brousseau, Roland; Coux, Florence; Frutos, Roger; Masson, Luke; Moozar, Kouros; Rang, Cecile; Rivest, Sebastien; Royer, Monique; Schwartz, Jean-Louis; Vachon, Vincent

Journal of Biological Chemistry, VOL. 276, NO. 38, September 21, 2001, PP. 35546-35551

The four salt bridges (Asp222-Arg281, Arg233-Glu288, Arg234-Glu274, and Asp242-Arg265) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp242-Arg265 bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

DESCRIPTOR(S)- *Membranes (Cell Biology); *Methods and Techniques; *Toxicology; *Bacillus thuringiensis (Endospore-forming Gram-Positives); *Manduca sexta (Lepidoptera) --larva; *Animals; *Arthropods; *Bacteria; *Eubacteria; *Insects; *Invertebrates; *Microorganisms; *midgut --digestive system; *trypsin --activation; *Cry1Aa --toxin; *D222A --alpha-helix 7 mutant; *D242A --alpha-helix 7 mutant; *D242E --toxin; *D242K --toxin; *D242N --toxin; *D242P --toxin; *R233A --alpha-helix 7 mutant; *R234A --alpha-helix 7 mutant; *light-scattering analysis --analytical method; *light-scattering analysis --Spectrum Analysis Techniques; *osmotic swelling assay --laboratory equipment; *Hi-Tech Scientific stopped-flow rapid kinetics apparatus --laboratory equipment; *Hi-Tech Scientific stopped-flow rapid kinetics apparatus --Hi-Tech Scientific; *Spex Fluorolog CM-3 spectrofluorometer --laboratory equipment; *interdomain salt bridge; *molecular flexibility; *pore formation; *proteolysis

2001

Rapid assay for avidin and biotin based on fluorescence quenching.

Song, Xuedong; Swanson, Basil I.

Analytica Chimica Acta, VOL. 442, NO. 1, 31 August, 2001, PP. 79-87

Biotin was covalently tagged with a BODIPY dye which can undergo an efficient distance-dependent fluorescence self-quenching. Multivalent binding of avidin with the BODIPY-labeled biotin (B581/591-biotin, either in aqueous buffer, or anchored on the surfaces of lipid vesicles or lipid bilayers coated on glass beads) induces aggregation of the BODIPY dye (up to four dyes for each avidin) to result in a decrease in fluorescence intensity due to fluorescence self-quenching. The system can be used to perform a rapid, direct assay for avidin and competitive assay for biotin with high sensitivity (<50 pM for avidin and <0.2 nM for biotin) and selectivity. The assay method is generally applicable for detection of all the species involved in a multivalent binding interaction.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *avidin --analysis; *avidin --multivalent binding; *avidin --rapid assay; *biotin --uses; *lipid bilayers; *lipid vesicles; *BODIPY dye --uses; *fluorescence self-quenching technique --applications; *fluorescence self-quenching technique --assay method; *fluorescence self-quenching technique --description; *fluorescence self-quenching technique --Bioassays/Physiological Analysis; *fluorescence self-quenching technique --Molecular Biology Techniques and Chemical Characterization; *spectrofluorometry --analytical method; *spectrofluorometry --Spectrum Analysis Techniques; *spectrophotometry --analytical method; *spectrophotometry --photometry: CB; *H-P 8452A diode array spectrophotometer --equipment; *H-P 8452A diode array spectrophotometer --Hewlett-Packard; *SPEX fluorolog-2 spectrofluorometer --equipment; *biotechnology; *molecular interactions --analysis; *molecular interactions --applications

2001

Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction. Optimization and application to single cell analysis.

Daunert, Sylvia; Feltus, Agatha; Hentz, Nathaniel G.

Journal of Chromatography A , VOL. 918, NO. 2, 25 May, 2001, PP. 381-392

A class-selective post-capillary reaction detection method for capillary electrophoresis is described in which a streptavidin-fluorescein isothiocyanate (streptavidin-FITC) conjugate is used to detect biotin moieties. The selective binding of biotin moieties to the streptavidin-FITC conjugate causes an enhancement of fluorescence proportional to the concentration of biotin present. After capillary electrophoresis the separated analytes react with streptavidin-FITC in a coaxial reactor and are then detected either by a benchtop spectrofluorometer (2.5 muM detection limit) or by an epi-fluorescence microscope (1.10-7 M detection limit). The method is used to examine biotinylated species in a crude mammalian cell lysate which was found to contain 83+-3 fmol in 3600 cell volumes. In addition, it is used to examine the uptake of biotin by individual sea urchin oocytes. The results indicate that, in the oocytes, biocytin is the prevalent form of biotin and its concentration varies widely between cells (mean=2+-2 muM).

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *sea urchin (Echinoidea); *CREF cell line (Muridae) --rat embryo fibroblasts; *Animals; *Chordates; *Echinoderms; *Invertebrates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates; *oocytes --reproductive system; *biocytin --Sigma; *biotin --Sigma; *biotin moieties --selective binding; *streptavidin --Vector Labs; *streptavidin-fluorescein isothiocyanate conjugate; *benchtop spectrofluorometer --laboratory equipment; *capillary electrophoresis --analytical method; *capillary electrophoresis --electrophoretic techniques; *epi-fluorescence microscope --laboratory equipment; *fluorescence flow cell --laboratory equipment; *fluorescence flow cell --NSG Precision Cells; *post-capillary reaction detection --analytical method; *post-capillary reaction detection --Electrophoretic Techniques; *single cell analysis --analytical method; *single cell analysis --Histological/Cytological and Culture Techniques; *Nikon Diaphot 200 inverted epi-fluorescence microscope --laboratory equipment; *Nikon Diaphot 200 inverted epi-fluorescence microscope --Nikon; *SPEX Fluorolog-2 spectrofluorometer --laboratory equipment; *SPEX Fluorolog-2 spectrofluorometer --SPEX Industries; *optimization; *streptavidin-biotin interaction

2001

pH-jump-induced folding and unfolding studies of barstar: Evidence for multiple folding and unfolding pathways.

Udgaonkar, Jayant B.; Rami, Bhadresh R.

Biochemistry, VOL. 40, NO. 50, December 18, 2001, PP. 15267-15279

Equilibrium and kinetic characterization of the high pH-induced unfolding transition of the small protein barstar have been carried out in the pH range 7-12. A mutant form of barstar, containing a single tryptophan, Trp 53, completely buried in the core of the native protein, has been used. It is shown that the protein undergoes reversible unfolding above pH 10. The pH 12 form (the D form) appears to be as unfolded as the form unfolded by 6 M guanidine hydrochloride (GdnHCl) at pH 7 (the U form): both forms have similar fluorescence and far-UV circular dichroism (CD) signals and have similar sizes, as determined by dynamic light scattering and size-exclusion chromatography. No residual structure is detected in the D form: addition of GdnHCl does not alter its fluorescence and far-UV CD properties. The fluorescence signal of Trp 53 has been used to monitor folding and unfolding kinetics. The kinetics of folding of the D form in the pH range 7-11 are complex and are described by four exponential processes, as are the kinetics of unfolding of the native state (N state) in the pH range 10.5-12. Each kinetic phase of folding decreases in rate with increase in pH from 7 to 10.85, and each kinetic phase of unfolding decreases in rate with decrease in pH from 12 to 10.85. At pH 10.85, the folding and unfolding rates for any particular kinetic phase are identical and minimal. The two slowest phases of folding and unfolding have identical kinetics whether measured by Trp 53 fluorescence or by mean residue ellipticity at 222 nm. Direct determination of the increase in the N state with time of folding at pH 7 and of the D form with time of unfolding at pH 12, by means of double-jump assays, show that between 85 and 95% of protein molecules fold or unfold via fast pathways between the two forms. The remaining 5-15% of protein molecules appear to fold or unfold via slower pathways, on which at least two intermediates accumulate. The mechanism of folding from the high pH-denatured D form is remarkably similar to the mechanism of folding from the urea or GdnHCl-denatured U form.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *barstar --analysis; *barstar --separation; *guanidine hydrochloride --reagent; *dynamic light scattering chromatography --separation method; *dynamic light scattering chromatography --Chromatographic Techniques; *far-UV circular dichroism --analytical method; *far-UV circular dichroism --Spectrum Analysis Techniques; *fluorescence circular dichroism --analytical method; *fluorescence circular dichroism --Spectrum Analysis Techniques; *size exclusion chromatography --liquid chromatography; *size exclusion chromatography --separation method; *J-700 spectropolarimeter --laboratory equipment; *J-700 spectropolarimeter --Jasco; *Spex DM 3000 Fluorolog spectrofluorimeter --laboratory equipment; *pH

2001

New method for evaluating fluorescence emission of drilling mud and mud additives

Qiu, Zheng-Song; Wang, Fu-Hua; Wang, Shu-Qi; Yan, Jie-Nian; Zhu, Jin-Zhi

Shiyou Daxue Xuebao, VOL. 25, NO. 4, 2001/August, PP. p 39-43+46

The fluorescence emission mechanism and some defects existing in conventional fluorescence evaluation methods are discussed. A new method for fluorescence evaluation on mud and mud additives is established on the basis of lots of investigation. The new method can avoid fluorescence quench caused by high concentration of measured fluid and personal error. It is especially suitable for fluorescence analysis of condensate oils, light oils and some mud additives with lower wavelength than 400 nm. Compared with the traditional methods, the new method is more accurate and trustworthy for determining the influencing extent of mud and mud additives on cuttings fluorologging 12 Refs.

2001

Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling.

Parker, Ian; LaFerla, Frank M.; Leissring, Malcolm A.; Marchant, Jonathan S.; Stutzmann, Grace E.

Nature Biotechnology , VOL. 19, NO. 7, July, 2001, PP. 645-649

DsRed, a recently cloned red fluorescent protein, has attracted great interest as an expression tracer and fusion partner for multicolor imaging. We report that three-photon excitation (lambda<760 nm) rapidly changes the fluorescence of DsRed from red to green when viewed subsequently by conventional (one-photon) epifluorescence. Mechanistically, three-photon excitation (lambda<760 nm) selectively bleaches the mature, red-emitting form of DsRed, thereby enhancing emission from the immature green form through reduction of fluorescence resonance energy transfer (FRET). The "greening" effect occurs in live mammalian cells at the cellular and subcellular levels, and the resultant color change persists for >30 h without affecting cell viability. This technique allows individual cells, organelles, and fusion proteins to be optically marked and has potential utility for studying cell lineage, organelle dynamics, and protein trafficking, as well as for selective retrieval of cells from a population. We describe optimal parameters to induce the color change of DsRed, and demonstrate applications that show the potential of this optical highlighter.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *CHO cell line (Cricetidae); *HEK 293 cell line (Hominidae); *3t3 cell line (Muridae); *Animals; *Chordates; *Humans; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Primates; *Rodents; *Vertebrates; *DsRED --color change; *DsRED --multiphoton; *DsRED --red fluorescent protein; *fluorescence resonance energy transfer --analytical method; *fluorescence resonance energy transfer --spectroscopic techniques: CB; *multicolor imaging --imaging method; *multicolor imaging --imaging techniques; * Fluorolog spectrometer --equipment; * Fluorolog spectrometer --Fluorolog; *Olympus BX50 microscope --equipment; *Olympus BX50 microscope --Olympus

2001

Membrane-anchoring interactions of M13 major coat protein.

Hemminga, Marcus A.; Meijer, Alexander B.; Spruijt, Ruud B.; Wolfs, Cor J. A. M.

Biochemistry, VOL. 40, NO. 30, July 31, 2001, PP. 8815-8820

The response to hydrophobic mismatch of membrane-bound M13 major coat protein is measured using site-directed fluorescence and ESR spectroscopy. For this purpose, we investigate the membrane-anchoring interactions of M13 coat protein in model systems consisting of phosphatidylcholine bilayers that vary in hydrophobic thickness. Mutant coat proteins are prepared with an AEDANS-labeled single cysteine residue in the hinge region of the protein or at the C-terminal side of the transmembrane helix. In addition, the fluorescence of the tryptophan residue is studied as a monitor for the N-terminal side of the transmembrane helix. The fluorescence results show that the hinge region and C-terminal side of the transmembrane helix hardly respond to hydrophobic mismatch. In contrast, the N-terminal side of the helical transmembrane domain shifts to a more apolar environment, when the hydrophobic thickness is increased. The apparent strong membrane-anchoring interactions of the C-terminus are confirmed using a mutant that contains a longer transmembrane domain. As a result of this mutation, the tryptophan residue at the N-terminal side of the helical domain clearly shifts to a more polar environment, whereas the labeled position 46 at the C-terminal side is not affected. The phenylalanines in the C-terminal part of the protein play an important role in these apparent strong anchoring interactions. This is demonstrated with a mutant in which both phenylalanines are replaced by alanine residues. The phenylalanine residues in the C-terminus affect the location in the membrane of the entire transmembrane domain of the protein.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *alanine; *phenylalanines; *phosphatidylcholine bilayers --hydrophobic thickness; *tryptophan residue --fluorescence; *M13 major coat protein --membrane-anchoring interactions; *M13 major coat protein --membrane-bound; *site-directed fluorescence --analytical method; *site-directed fluorescence --Spectrum Analysis Techniques; *Bruker ESP 300E ESR spectrometer --laboratory equipment; *Bruker ESP 300E ESR spectrometer --Bruker; *ESR spectroscopy --analytical method; *ESR spectroscopy --spectroscopic techniques: CB; *SPEX Fluorolog 3-22 fluorometer --laboratory equipment; *hydrophobic mismatch --response

2001

Mapping the antagonist binding site of the serotonin type 3 receptor by fluorescence resonance energy transfer.

Vogel, Horst; Friedrich-Benet, Kirstin; Hovius, Ruud; Pick, Horst; Tairi, Ana-Paula; Vallotton, Pascal; Wohland, Thorsten

Biochemistry, VOL. 40, NO. 41, October 16, 2001, PP. 12237-12242

We have measured fluorescence resonance energy transfer (FRET) between a fluorescent antagonist, bound to the purified detergent-solubilized serotonin type 3 receptor, and a lipophilic acceptor probe partitioned into the micelle surrounding the detergent-solubilized receptor. The experimentally observed FRET efficiency was evaluated on the basis of the characteristic dimensions of the receptor-micelle complex and the average number of acceptor molecules in such micelles. The binding site was determined to be 5.4+-0.9 nm above the center of the detergent micelle. The experiments were performed below the critical micellar concentration of the detergent (C12E9) used to solubilize the receptor, under which conditions it was demonstrated that the ligand binding activity was fully preserved. This reduces considerably the fluorescence background arising from probes not associated with the receptor, allowing a precise determination of the transfer efficiency.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Pharmacology; *detergents --uses; *fluorescent antagonist --uses; *ligands; *micelles; *molecular probes; *proteins; *serotonin; *serotonin receptors --analysis; *serotonin receptors --functions; *serotonin receptors --structures; *serotonin type 3 receptors --analysis; *serotonin type 3 receptors --antagonist binding site mapping methodologies; *serotonin type 3 receptors --functions; *serotonin type 3 receptors --purified detergent-solubilized; *serotonin type 3 receptors --structures; *chromatography --purification method; *chromatography --Chromatographic Techniques; *fluorescence resonance energy transfer technique --analytical method; *fluorescence resonance energy transfer technique --applications; *fluorescence resonance energy transfer technique --description; *fluorescence resonance energy transfer technique --molecular method; *fluorescence resonance energy transfer technique --Molecular Biology Techniques and Chemical Characterization; *spectrophotometry --analytical method; *spectrophotometry --photometry; *Beckman DU7500i spectrophotometer --equipment; *Beckman DU7500i spectrophotometer --Beckman; *Beckman DU7500i spectrophotometer --Coulter; *SPEX Fluorolog II --equipment; *SPEX Fluorolog II --Instruments S. A.; *methodology; *molecular models

2001

Intra- and intermolecular beta-pleated sheet formation in glutamine-repeat inserted myoglobin as a model for polyglutamine diseases.

Nukina, Nobuyuki; Akagi, Takumi; Hashikawa, Tsutomu; Morishima, Isao; Tanaka, Motomasa

Journal of Biological Chemistry, VOL. 276, NO. 48, November 30, 2001, PP. 45470-45475

An aberrant structure of the expanded polyglutamine might be involved in the formation of aggregates in CAG repeat diseases. To elucidate structural properties of the expanded polyglutamine, we prepared sperm whale myoglobin (Mb) mutants, in which 12, 28, 35, and 50 repeats of glutamine were inserted at the corner between the C and D helices (Gln12, Gln28, Gln35, and Gln50, respectively). Circular dichroism and IR spectroscopies showed that the expanded polyglutamine, which was recognized by the monoclonal antibody 1C2 in Gln28, Gln35, and Gln50 Mb forms an antiparallel beta-pleated sheet structure. Gln50 Mb aggregates were found to comprise an intermolecular antiparallel beta-pleated sheet. Fluorescence together with 1H NMR spectra revealed partial unfolding of the protein surface in Gln35 and Gln50 Mb, although the structural changes in the protein core were rather small. The present results indicate that the fluctuating beta-pleated sheet of the expanded polyglutamine exposed on the protein surface facilitates the formation of aggregates through intermolecular interactions. The present study has first established and characterized structural properties of a molecular model for polyglutamine diseases in which various lengths of polyglutamine including a pathologically expanded glutamine repeat were inserted into a structurally known protein.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *sperm whale (Physeteridae); *Animals; *Cetaceans; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Vertebrates; *polyglutamine disease --nervous system disease; *glutamine; *myoglobin --glutamine-repeat inserted; *myoglobin --intermolecular beta-pleated sheet formation; *myoglobin --intramolecular beta-pleated sheet formation; *polyglutamine --analysis; *1C2 --monoclonal antibody; *1C2 --Chemicon; *circular dichroism --analytical method; *circular dichroism --Spectrum Analysis Techniques; *fluorescence --analytical method; *fluorescence --Spectrum Analysis Techniques; *proton NMR --analytical method; *proton NMR --Spectrum Analysis Techniques; *BRUKER Avance DRX600 spectrometer --laboratory equipment; *BRUKER Avance DRX600 spectrometer --BRUKER; * Fluorolog spectrofluorometer --laboratory equipment; * Fluorolog spectrofluorometer --HORIBA; *FT-IR 8300 spectrophotometer Fourier transform IR 8300 spectrophotometer --laboratory equipment; *FT-IR 8300 spectrophotometer Fourier transform IR 8300 spectrophotometer --Shimadzu; *IR spectroscopy --analytical method; *IR spectroscopy --Spectrum Analysis

2001

Integrated Instrumentation System for the Characterization of Polymer Solution and Gel-Light-Emitting Devices

Yang, Yang

2001-03-31, California Univ., US Academic Los Angeles, CA United States

This DURIP grant has contributed strongly in expanding my research activity at UCLA. Being a young professor with limited funding for equipment, this DURIP grant helped me to equip my laboratory and allow me to expand my research activity into different areas, such as the study of nano-scale polymer morphology by using the atomic force microscope. Compared to the originally proposed equipment list, there have been some modifications in the purchased equipment. Our original budget includes a Hewlett-Packard GPC, a Fluorolog 3 spectrometer, an AC impedance meter, a Bio-Analytical Electrochemical Station. However, we later realized that Prof. Fred Wudl, my collaborator on the polymer SLED project, has the HP GPC, AC Impedance meter, and Bio-Analytical Electrochemical Station in his laboratory. He welcomes my group to use those equipments. I consider the best way of using the DURIP funds is to purchase important equipment, and not to duplicate the existing equipment. With the permission from AFOSR (Please see attached document), we used the DURIP funds that we budgeted for the Bio-Analytical station, AC Impedance meter, and GPC to acquire an Atomic Force Microscope (AFM from Digital Instrument Nano-3A) and a Nikon SMZ 1500 Stereo Microscope. The total amount of the purchased equipment is $194,086. Among them, $112,063 is from AFOSR's DURIP, and the rest of $82,023 is matching funds from UCLA and SONY Corporation.
PROGRESS- Final Report, 1 Apr. 2000-31 Mar. 2001

2001

Glucose-induced oscillations in cytoplasmic free Ca2+ concentration precede oscillations in mitochondrial membrane potential in the pancreatic beta-cell.

Kindmark, Henrik; Berggren, Per-Olof; Branstrom, Robert; Brown, Graham; Kohler, Martin; Larsson, Olof

Journal of Biological Chemistry, VOL. 276, NO. 37, September 14, 2001, PP. 34530-34536

Using dual excitation and fixed emission fluorescence microscopy, we were able to measure changes in cytoplasmic free Ca2+ concentration ((Ca2+)i) and mitochondrial membrane potential simultaneously in the pancreatic beta-cell. The beta-cells were exposed to a combination of the Ca2+ indicator fura-2/AM and the indicator of mitochondrial membrane potential, rhodamine 123 (Rh123). Using simultaneous measurements of mitochondrial membrane potential and (Ca2+)i during glucose stimulation, it was possible to measure the time lag between the onset of mitochondrial hyperpolarization and changes in (Ca2+)i. Glucose-induced oscillations in (Ca2+)i were followed by transient depolarizations of mitochondrial membrane potential. These results are compatible with a model in which nadirs in (Ca2+)i oscillations are generated by a transient, Ca2+-induced inhibition of mitochondrial metabolism resulting in a temporary fall in the cytoplasmic ATP/ADP ratio, opening of plasma membrane KATP channels, repolarization of the plasma membrane, and thus transient closure of voltage-gated L-type Ca2+ channels.

DESCRIPTOR(S)- *Endocrine System (Chemical Coordination and Homeostasis); *Membranes (Cell Biology); *mouse (Muridae) --adult; *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates; *mitochondria; *pancreatic beta-cells --endocrine system; *plasma membrane --repolarization; *calcium --cytoplasmic free concentration; *fura-2/AM --calcium indicator; *fura-2/AM --Molecular Probes; *glucose; *potassium channel; *rhodamine 123 --cellular distribution; *rhodamine 123 --Molecular Probes; *confocal laser scanning microscope system --equipment; *confocal laser scanning microscope system --Leica; *fixed emission fluorescence microscopy --microscopy method; *fixed emission fluorescence microscopy --microscopy: CB; *Leica DMIRB microscope --equipment; *Leica DMIRB microscope --Leica; *SPEX fluorolog-2 CM1T11I system --equipment; *SPEX fluorolog-2 CM1T11I system --Zeiss; *calcium oscillations; *dual excitation; *mitochondrial membrane potential --transient depolarizations; *ATP/ADP ratio
BIOLOGICAL TAXONOMIC DESCRIPTOR(S)- Muridae --Animalia; Muridae --Chordata; Muridae --Mammalia; Muridae --Rodentia; Muridae --Vertebrata

2001

Formation of soluble inclusion bodies by HPV E6 oncoprotein fused to maltose-binding protein.

Trave, Gilles; Laurent, Cecile; Lefevre, Jean-Francois; Nomine, Yves; Ristriani, Tutik; Weiss, Etienne

Protein Expression and Purification, VOL. 23, NO. 1, October, 2001, PP. 22-32

Many polypeptides overexpressed in bacteria are produced misfolded and accumulate as solid structures called inclusion bodies. Inclusion-body-prone proteins have often been reported to escape precipitation when fused to maltose-binding protein (MBP). Here, we have examined the case of HPV 16 oncoprotein E6. The unfused sequence of E6 is overexpressed as inclusion bodies in bacteria. By contrast, fusions of E6 to the C-terminus of MBP are produced soluble. We have analyzed preparations of soluble MBP-E6 fusions by using three independent approaches: dynamic light scattering, lateral turbidimetry, and sandwich ELISA. All three methods showed that MBP-E6 preparations contain highly aggregated material. The behavior of these soluble aggregates under denaturating conditions suggests that they are formed by agglomeration of misfolded E6 moieties. However, precipitation is prevented by the presence of the folded and highly soluble MBP moieties, which maintain the aggregates in solution. Therefore, the fact that a protein or protein domain is produced soluble when fused to the C-terminus of a carrier protein does not guarantee that the protein of interest is properly folded and active. We suggest that aggregation of fusion proteins should be systematically assayed, especially when these fusions are to be used for binding measurements or activity tests.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Escherichia coli (Enterobacteriaceae) --strain-XL1Blue; *HPV human papillomavirus (Papovaviridae) --pathogen; *Animal Viruses; *Bacteria; *Eubacteria; *Microorganisms; *Viruses; *maltose-binding protein --carboxy-terminus; *maltose-binding protein-E6 fusions --analysis; *E6 oncoprotein --expression; *dynamic light scattering --analytical method; *dynamic light scattering --Molecular Biology Techniques and Chemical Characterization; *lateral turbidimetry --analytical method; *lateral turbidimetry --densitometry; *microvinyl plates --laboratory equipment; *microvinyl plates --Falcon; *photon counting photomultiplier --laboratory equipment; *sandwich ELISA --analytical method; *sandwich ELISA --labeling; *ELISA reader --laboratory equipment; *LS-230 instrument --laboratory equipment; *LS-230 instrument --Beckman-Coulter; *SPEX Fluorolog-2 spectrofluorometer --laboratory equipment; *SPEX Fluorolog-2 spectrofluorometer --Inc.; *SPEX Fluorolog-2 spectrofluorometer --SPEX Industries; *denaturating conditions; *soluble inclusion bodies --formation

2001

Fluorescence-based analyses of the effects of full-length recombinant TAF130p on the interaction of TATA box-binding protein with TATA box DNA.

Weil, P. Anthony; Banik, Utpal; Beechem, Joseph M.; Klebanow, Edward; Schroeder, Stephanie

Journal of Biological Chemistry, VOL. 276, NO. 52, December 28, 2001, PP. 49100-49109

We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae) --expression system; *Saccharomyces cerevisiae (Ascomycetes); *Bacteria; *Eubacteria; *Fungi; *Microorganisms; *Nonvascular Plants; *Plants; *recombinant TAF130p --analysis; *recombinant TAF130p --full-length; *TATA box DNA --analysis; *TATA box-binding protein TBP --analysis; *fluorescence anisotropy spectroscopy --analytical method; *fluorescence anisotropy spectroscopy --Spectrum Analysis Techniques; *fluorescence-based native gel electrophoresis --analytical method; *fluorescence-based native gel electrophoresis --Electrophoretic Techniques; *T-format SPEX 1681 Fluorolog spectrofluorimeter --laboratory equipment; *protein-DNA interaction

2001

Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein.

Vogel, Horst; Cattarinussi, Serge; Eisenhawer, Martin; Kuhn, Andreas

Biochemistry, VOL. 40, NO. 41, October 16, 2001, PP. 12321-12328

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *phage M13 (Inoviridae); *Bacterial Viruses; *Microorganisms; *Viruses; *artificial lipid bilayers --applications; *membrane proteins --folding mechanisms; *membrane proteins --molecular analysis; *membrane proteins --molecular topology; *phage coat proteins --functions; *phage coat proteins --molecular analysis; *phage coat proteins --structures; *phage transmembrane procoat protein --close helix-helix distance; *phage transmembrane procoat protein --fluorescence resonance energy transfer; *phage transmembrane procoat protein --functions; *phage transmembrane procoat protein --molecular analysis; *phage transmembrane procoat protein --structures; *fluorescence resonance energy transfer measurement technique --analytical method; *fluorescence resonance energy transfer measurement technique --applications; *fluorescence resonance energy transfer measurement technique --Molecular Biology Techniques and Chemical Characterization; *Spex Fluorolog fluorometer --equipment; *Spex Fluorolog fluorometer --uses; *gene mutations; *molecular models

2001

Fesselin, a synaptopodin-like protein, stimulates actin nucleation and polymerization.

Chalovich, Joseph M.; Beall, Brent

Biochemistry, VOL. 40, NO. 47, November 27, 2001, PP. 14252-14259

Fesselin is a proline-rich actin binding protein that has recently been isolated from smooth muscle (Leinweber, B. D., Fredricksen, R. S., Hoffman, D. R., and Chalovich, J. M. (1999) J. Muscle Res. Cell Motil. 20, 539-545). Fesselin is similar to synaptopodin (Mundel, P., Heid, H. W., Mundel, T. M., Kruger, M., Reiser, J., and Kriz, W. (1997) J. Cell Biol. 139, 193-204) in terms of its size, isoelectric point, and sequence although synaptopodin is not present in smooth muscle. The function of fesselin is unknown. Evidence is presented here that fesselin accelerates the polymerization of actin. Fesselin was effective on actin isolated from either smooth or skeletal muscle at low ionic strength and in the presence of 100 mM KCl. At low ionic strength, fesselin decreased the time for 50% polymerization to about 1% of that in the absence of fesselin. The lag phase characteristic of the slow nucleation process of polymerization was eliminated as the fesselin concentration was increased from very low levels. Fesselin did not alter the critical concentration for actin but did increase the rate of elongation by apprxeq3-fold. The increase in elongation rate constant is insufficient to account for the total increase in polymerization rate. It is likely that fesselin stabilizes the formation of actin nuclei. Time courses of actin polymerization at varied fesselin concentrations and varied actin concentrations were simulated by increasing the rate of nucleation and both the forward and reverse rate constants for elongation.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Muscular System (Movement and Support); *rabbit (Leporidae); *Animals; *Chordates; *Lagomorphs; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Vertebrates; *skeletal muscle --muscular system; *smooth muscle --muscular system; *actin --nucleation; *actin --polymerization; *fesselin --synaptopodin-like protein; *potassium chloride; *gel-filtration chromatography --purification method; *gel-filtration chromatography --Chromatographic Techniques; *polymerization assays --analytical method; *polymerization assays --Molecular Biology Techniques and Chemical Characterization; *Applied Photophysics DX17.MV/2 sequential mixing stopped-flow spectrophotometer --laboratory equipment; *Applied Photophysics DX17.MV/2 sequential mixing stopped-flow spectrophotometer --Applied Photophysics; * Fluorolog 2 spectrofluorometer --laboratory equipment; * Fluorolog 2 spectrofluorometer --Spex; *Sephacryl S-200 column --laboratory equipment; *Sephacryl S-200 column --Pharmacia LKB Biotechnology Inc.; *elongation --forward rate constant; *elongation --reverse rate constant

2001

Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis.

Niederweis, Michael; Ehrt, Sabine; Kaps, Iris; Martin, Carlos; Riley, Lee W.; Schnappinger, Dirk; Seeber, Silke

Gene (Amsterdam) , VOL. 278, NO. 1-2, 31 October, 2001, PP. 115-124

The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis. Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP). A 160-fold variation of GFP expression levels in M. smegmatis was achieved by combining three promoters with different copy numbers of the vectors. An efficient energy transfer between BFP and GFP in M. smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors. Thus, these vectors will be valuable for all strategies where co-expression of proteins in M. smegmatis is needed, e.g. for constructing a two-hybrid system or for deleting essential genes.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Mycobacterium smegmatis (Mycobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *blue fluorescent protein BFP --analysis; *blue fluorescent protein BFP --fluorescent protein; *green fluorescent protein GFP --analysis; *green fluorescent protein GFP --fluorescent protein; *plasmid vectors --gene vector; *plasmid vectors --synthesis; *charged-coupled device camera Axiophot 2 --laboratory equipment; *charged-coupled device camera Axiophot 2 --Zeiss; *fluorescence microscope Axioplan 2 --laboratory equipment; *fluorescence microscope Axioplan 2 --Zeiss; *fluorescence resonance energy transfer FRET --analytical method; *fluorescence resonance energy transfer FRET --Spectrum Analysis Techniques; *two-plasmid co-expression system --laboratory equipment; * Fluorolog-3 --laboratory equipment; * Fluorolog-3 --ISA; *energy transfer

2001

Effect of urea denaturation on tryptophan fluorescence and nucleotide binding on tubulin studied by fluorescence and NMR spectroscopic methods.

Kasturi, S. R.; Kuchroo, K.; Maity, H.

Physiological Chemistry and Physics and Medical NMR, VOL. 33, NO. 2, 2001, PP. 139-151

Tubulin, the major protein of microtubules, has been shown to be an example of a protein undergoing multistep unfolding. Local unfolding and stepwise loss of a number of characteristic functions were demonstrated. In order to understand urea induced effects on tryptophan fluorescence and nucleotide binding on tubulin, both fluorescence and NMR techniques were used. Tubulin was denatured by different urea concentrations. The present experiments were carried out at concentrations of tubulin (apprx10 muM) at which most of the protein will be in the dimeric state. Quenching studies in the presence of KI suggest that all the tryptophans are fairly solvent exposed. Similar studies using acrylamide as quencher, suggest unfolding of tubulin at these protein concentrations to be an apparent two state process between the native and the completely unfolded states unlike at low concentrations where a partially folded intermediate was observed. No observable effects of the nucleotide or the metal ion on tryptophan fluorescence were observed. An attempt was made using NMR to monitor the changes in the nucleotide interaction with tubulin as the protein is unfolded by urea denaturation. No significant effects were observed in the binding of the nucleotide to tubulin by urea denaturation.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *nucleotide --binding; *tryptophan --fluorescence; *tubulin --analysis; *urea; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *Spex Fluorolog model 168IT spectrofluorimeter --laboratory equipment; *Unity plus 600 MHz spectrometer --laboratory equipment; *Unity plus 600 MHz spectrometer --Varian

2001

DNA interaction of the tyrosine protein kinase inhibitor PD153035 and its N-methyl analogue.

Bailly, Christian; Baldeyrou, Brigitte; Bouey-Bencteux, Edith; Colson, Pierre; Goossens, Jean-Francois; Henichart, Jean-Pierre; Houssier, Claude; Houssin, Raymond; Laine, William

Biochemistry, VOL. 40, NO. 15, April 17, 2001, PP. 4663-4671

The brominated anilinoquinazoline derivative PD153035 exhibits a very high affinity and selectivity for the epidermal growth factor receptor tyrosine kinase (EGF-R TK) and shows a remarkable cytotoxicity against several types of tumor cell lines. In contrast, its N-methyl derivative, designated EBE-A22, has no effect on EGF-R TK but maintains a high cytotoxic profile. The present study was performed to explore the possibility that PD153035 and its N-methyl analogue might interact with double-stranded DNA, which is a primary target for many conventional antitumor agents. We studied the strength and mode of binding to DNA of PD153035 and EBE-A22 by means of absorption, fluorescence, and circular and linear dichroism as well as by a relaxation assay using human DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to show that both drugs bind to DNA and behave as typical intercalating agents. In particular, EBE-A22 unwinds supercoiled plasmid, stabilizes duplex DNA against heat denaturation, and produces negative CD and ELD signals, as expected for an intercalating agent. Extensive DNase I footprinting experiments performed with a large range of DNA substrates show that EBE-A22, but not PD153035, interacts preferentially with GC-rich sequences and discriminates against homooligomeric runs of A and T which are often cut more readily by the enzyme in the presence of the drug compared to the control. Altogether, the results provide the first experimental evidence that DNA is a target of anilinoquinazoline derivatives and suggest that this N-methylated ring system is a valid candidate for the development of DNA-targeted cytotoxic compounds. The possible relevance of selective DNA binding to activity is considered. The unexpected GC-selective binding properties of EBE-A22 entreat further exploration into the use of N-methylanilinoquinazoline derivatives as tools for designing sequence-specific DNA binding ligands.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *DNA --interaction; *DNA binding ligands --sequence-specific; *N-methyl analogue; *N-methylanilinoquinazoline derivatives; *PD153035 --tyrosine protein kinase inhibitor; *absorption assay --analytical method; *absorption assay --Analysis/Characterization Techniques: CB; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *fluorescence assay --analytical method; *fluorescence assay --Analysis/Characterization Techniques: CB; *gel electrophoresis --analytical method; *gel electrophoresis --electrophoretic techniques; *linear dichroism --analytical method; *linear dichroism --spectroscopic techniques: CB; *optical technique --analytical method; *optical technique --Analysis/Characterization Techniques: CB; *relaxation assay --analytical method; *relaxation assay --Analysis/Characterization Techniques: CB; *DNase I footprinting --analytical method; *DNase I footprinting --DNA footprinting; *Jobin-Yvon CD 6 dichrograph --laboratory equipment; *Jobin-Yvon CD 6 dichrograph --Jobin-Yvon; *Molecular Dynamics 425E PhosphorImager --laboratory equipment; *Molecular Dynamics 425E PhosphorImager --Molecular Dynamics; *SPEX fluorometer Fluorolog --laboratory equipment; *SPEX fluorometer Fluorolog --SPEX; *Uvikon 943 spectrophotometer --laboratory equipment; *Uvikon 943 spectrophotometer --Uvikon

2001

Conformational equilibrium of the reactive center loop of antithrombin examined by steady state and time-resolved fluorescence measurements: Consequences for the mechanism of factor Xa inhibition by antithrombin-heparin complexes.

Gettins, Peter G. W.; Beechem, Joseph M.; Futamura, Akiko

Biochemistry, VOL. 40, NO. 22, June 5, 2001, PP. 6680-6687

Activation of antithrombin by high-affinity heparin as an inhibitor of factor Xa has been ascribed to an allosteric switch between two conformations of the reactive center loop. However, we have previously shown that other, weaker binding, charged polysaccharides can give intermediate degrees of activation (Gettins, P. G. W., et al. (1993) Biochemistry 32, 8385-8389). To examine whether such intermediate activation results from different reactive center loop conformations or, more simply, from a different equilibrium constant between the same two extreme conformations, we have used NBD covalently bound at the P1 position of an engineered R393C variant of antithrombin as a fluorescent reporter group and measured fluorescence lifetimes of the label in free antithrombin as well as in antithrombin saturated with long-chain high-affinity heparin, high-affinity heparin pentasaccharide, long-chain low-affinity heparin, and dextran sulfate. Steady state emission spectra, anisotropies, and dynamic quenching measurements were also recorded. We found that the large steady state fluorescence enhancements produced by binding of activators resulted from relief of a static quench of fluorescence of NBD in apprx50% of the labeled antithrombin molecules rather than from any large change in lifetimes, and that similar lifetimes were found for NBD in all activated antithrombin-oligosaccharide complexes. Similar anisotropies and positions of the NBD emission maxima were also found in the absence and presence of activators. In addition, NBD was accessible to quenching agents in both the absence and presence of activators, with an at most 2-fold increase in quenching constants between these two extremes. The simplest interpretation of the partial static quench in the absence of activators, the different degrees of enhancement by different antithrombin activators, and the similar fluorescence properties and quenching behavior of the different states is that there are two distinct types of conformational equilibrium involving three distinct states of antithrombin, which we designate A, A', and B. A' represent low-affinity or inactivate states of approximately equal energy, both having the hinge residues inserted into beta-sheet A. A is fluorescent, while A' is statically quenched. State B represents the activated loop-expelled conformation in which none of the NBD fluorophores are statically quenched, as a result of the loop, including the P1-NBD, moving away from the body of the antithrombin. Different activators are able to shift the equilibrium to the high-activity (B) state to different extents and hence give different degrees of measured activity, and different degrees of relief of static quench. The similar properties and accessibility of the NBD in the A and B conformations also indicate that the P1 side chain is not buried in the low-activity A conformation, suggesting that an earlier proposal that activation involves exposure of the P1 side chain cannot be the explanation for activation. As an alternative explanation, heparin activation may give access to an exosite on antithrombin for binding to factor Xa and hence be the principal basis for enhancement of the rate of inhibition.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *antithrombin --activation; *antithrombin --conformational equilibrium; *antithrombin --reactive center loop; *factor Xa; *high-affinity heparin; *NBD fluorophores; *steady state fluorescence --analytical method; *steady state fluorescence --Analysis/Characterization Techniques: CB; *time-resolved fluorescence --analytical method; *time-resolved fluorescence --Analysis/Characterization Techniques: CB; *SLM 8000 spectrofluorometer --equipment; *SPEX Fluorolog 2 fluorometer --equipment

2001

Conformational changes that occur during an RNA-editing adenosine deamination reaction.

Beal, Peter A.; Stephens, Olen M.; Yi-Brunozzi, Hye Young

Journal of Biological Chemistry, VOL. 276, NO. 41, October 12, 2001, PP.37827-37833

ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2cntdotRNA complex.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *adenosine deaminase 2 ADAR2 --RNA binding domain; *adenosine deaminases; *duplex RNA; *nucleotides --conformational flexibility; *tryptophan --fluorescence; *2-aminopurine --fluorescence; *acrylamide quenching experiments --analytical method; *acrylamide quenching experiments --Molecular Biology Techniques and Chemical Characterization; *electrophoresis --purification method; *electrophoresis --Electrophoretic Techniques; *fluorescence --analytical method; *fluorescence --Spectrum Analysis Techniques; *hydroxyl radical footprinting --analytical method; *hydroxyl radical footprinting --Molecular Biology Techniques and Chemical Characterization; *storage phosphorimaging plates --laboratory equipment; *Instruments S. A., Inc., Fluorolog-3 spectrophotometer --laboratory equipment; *Instruments S. A., Inc., Fluorolog-3 spectrophotometer --Inc.; *Instruments S. A., Inc., Fluorolog-3 spectrophotometer --Instruments S. A.; *adenosine deaminase 2-RNA complex ADAR2-RNA complex ; *conformational changes; *protein-RNA interactions; *site-selective adenosine deamination; *RNA-editing adenosine deamination reaction

2001

Configurations of the N-terminal amphipathic domain of the membrane-bound M13 major coat protein.

Hemminga, Marcus A.; Meijer, Alexander B.; Spruijt, Ruud B.; Wolfs, Cor J. A. M.

Biochemistry , VOL. 40, NO. 16, April 24, 2001, PP. 5081-5086

The M13 major coat protein has been extensively studied in detergent-based and phospholipid model systems to elucidate its structure. This resulted in an L-shaped model structure of the protein in membranes. An amphipathic alpha-helical N-terminal arm, which is parallel to the surface of the membrane, is connected via a flexible linker to an alpha-helical transmembrane domain. In the present study, a fluorescence polarity probe or ESR spin probe is attached to the SH group of a series of N-terminal single cysteine mutants, which were reconstituted into DOPC model membranes. With ESR spectroscopy, we measured the local mobility of N-terminal positions of the protein in the membrane. This is supplemented with relative depth measurements at these positions by fluorescence spectroscopy via the wavelength of maximum emission and fluorescence quenching. Results show the existence of at least two possible configurations of the M13 amphipathic N-terminal arm on the ESR time scale. The arm is bound either to the membrane surface or in the water phase. The removal or addition of a hydrophobic membrane-anchor by site-specific mutagenisis changes the ratio between the membrane-bound and the water phase fraction.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *M13 --major coat protein; *M13 --membrane bound; *M13 --N-terminal amphipathic domain; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *site-specific mutagenesis --analytical method; *site-specific mutagenesis --Molecular Biology Techniques and Chemical Characterization; *Bruker ESP 300E ESR spectrometer --laboratory equipment; *Bruker ESP 300E ESR spectrometer --Bruker; *ESR spectroscopy --analytical method; *ESR spectroscopy --spectroscopic techniques: CB; *ESR spectroscopy --spectroscopic techniques: CT; *SPEX Fluorolog 3-22 fluorometer --laboratory equipment; *SPEX Fluorolog 3-22 fluorometer --SPEX; *fluorescence polarity probe; *L-shaped structure model

2001

Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3.

Low, Philip S.; Zhou, Jianzhong

Journal of Biological Chemistry, VOL. 276, NO. 41, October 12, 2001, PP.38147-38151

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 ANG) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *ankyrin; *erythrocyte membrane band 3 cdb3 --characterization; *erythrocyte membrane band 3 cdb3 --cytoplasmic domain; *erythrocyte membrane band 3 cdb3 --dimer; *erythrocyte membrane band 3 cdb3 --reversible conformational equilibrium; *flanking peripheral protein binding domains; *shared dimerization domain; *urea --stability; *D316A --acceptor residue; *W105L --donor residue; *crystallography --crystallization method; *crystallography --Crystallographic Techniques; * Fluorolog-3 SPEX spectrofluorimeter --laboratory equipment; * Fluorolog-3 SPEX spectrofluorimeter --Inc.; * Fluorolog-3 SPEX spectrofluorimeter --Instruments S.A.; *global membrane properties; *intrinsic fluorescence; *segmental mobility; *structural equilibrium; *thermal stability; *Stokes radius

2001

Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex.

Hunter, C. Neil; Karger, Guy A.; Reid, James D.

Biochemistry, VOL. 40, NO. 31, August 7, 2001, PP. 9291-9299

Magnesium protoporphyrin chelatase catalyzes the insertion of a Mg2+ ion into protoporphyrin IX, which can be considered as the first committed step of (bacterio)chlorophyll synthesis. In the present work, the Mg chelatase H subunits from both Synechocystis and Rhodobacter sphaeroides were studied because of the differing requirements of these organisms for modified cyclic tetrapyrroles. Deuteroporphyrin was shown to be a substrate for Mg chelatase. Analytical HPLC gel filtration was used to show that an H-deuteroporphyrin complex can be reconstituted by incubating the magnesium chelatase H subunit with a molar excess of deuteroporphyrin and that these complexes are monomers. The binding process occurs in the absence of Mg2+ or ATP or the I or D subunits of Mg chelatase. The emission from Trp residues in the H subunit is partly quenched when deuteroporphyrin is bound. Quantitative analysis of Trp fluorescence quenching led to determination of the Kd values for deuteroporphyrin binding to BchH from Rb. sphaeroides and ChlH from Synechocystis, which are 1.22+-0.42 muM and 0.53+-0.12 muM for ChlH and BchH, respectively. In the case of ChlH, but not BchH, the Kd increased 4-fold in the presence of MgATP2-. Red shifts in absorbance and excitation peaks were observed in the B band of the bound porphyrin in comparison with deuteroporphyrin in solution, as well as reduced yield and red shifts of up to 8 nm in fluorescence emission. These alterations are consistent with a slightly deformed nonplanar conformation of the bound porphyrin. Mg deuteroporphyrin, the product of the Mg chelation reaction, was shown to form a complex with either ChlH or BchH; in each case the Kd for Mg deuteroporphyrin is similar to that for deuteroporphyrin. The implications of the H-Mg protoporphyrin interaction for the next enzyme in the chlorophyll biosynthetic pathway, Mg protoporphyrin methyltransferase, are discussed.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Rhodobacter sphaeroides (Purple Nonsulfur Bacteria); *Synechocystis (Chroococcales); *Bacteria; *Cyanobacteria; *Eubacteria; *Microorganisms; *chlorophyll --synthesis; *cyclic tetrapyrroles; *deuteroporphyrin IX --binding properties; *deuteroporphyrin IX --characterization; *deuteroporphyrin IX --enzyme substrate; *magnesium chelatase --activity; *magnesium chelatase --H subunit; *magnesium protoporphyrin methyltransferase; *H-deuteroporphyrin complex --spectroscopic properties; *analytical HPLC gel filtration analytical high performance liquid chromatography gel filtration --liquid chromatography; *analytical HPLC gel filtration analytical high performance liquid chromatography gel filtration --purification method; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopy: CB; *tryptophan fluorescence quenching --analytical method; *tryptophan fluorescence quenching --Analysis/Characterization Techniques: CB; *SPEX Fluorolog spectrofluorometer --equipment; *porphyrin complex; *protein-protein interaction

2001

Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer.

Miller, Laurence J.; Cheng, Zhi-Jie

Journal of Biological Chemistry, VOL. 276 , NO. 51, December 21, 2001, PP. 48040-48047

Dimerization of some G protein-coupled receptors has recently been demonstrated, but how widespread this phenomenon might be and its functional implications are not yet clear. We have utilized biophysical and biochemical techniques to evaluate whether the type A cholecystokinin (CCK) receptor can form oligomeric complexes in the plasma membrane and the impact of ligand binding and signaling on such complexes. We investigated the possibility of bioluminescence resonance energy transfer (BRET) between receptor constructs that included carboxyl-terminal tags of Renilla luciferase or yellow fluorescent protein. Indeed, co-expression of these constructs in COS cells resulted in the constitutive presence of a significant BRET signal above that in a series of controls, with this signal reduced by co-expression of competing non-tagged CCK receptors. The presence of an oligomeric complex of CCK receptor molecules was confirmed in co-immunoprecipitation experiments. Occupation of CCK receptors with agonist ligands (CCK or gastrin-4) resulted in the rapid reduction in BRET signal in contrast to the enhancement of such a signal reported after agonist occupation of the beta2-adrenergic receptor. These effects on CCK receptor oligomerization were concentration-dependent, correlating with the potencies of the agonists. A smaller effect was observed for a partial agonist, and no effect was observed for antagonist occupation of this receptor. Agonist-induced reduction in BRET signal was also observed for pairs of CCK receptors with a donor-acceptor pair situated in other positions within the receptor. Manipulation of the phosphorylation state of CCK receptor using protein kinase C activation with phorbol ester or inhibition with staurosporine had no effect on the basal level or agonist effect on CCK receptor oligomerization. This provides the first evidence for CCK receptor oligomerization in living cells, with insights that the active conformation of this receptor dissociates these complexes in a phosphorylation-independent manner.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Cell Biology; *Endocrine System (Chemical Coordination and Homeostasis); *Methods and Techniques; *animal (Animalia); *rat (Muridae); *COS-1 cell line (Cercopithecidae); *Renilla (Cnidaria); *Animals; *Chordates; *Invertebrates; *Mammals; *Nonhuman Mammals; *Nonhuman Primates; *Nonhuman Vertebrates; *Primates; *Rodents; *Vertebrates; *plasma membranes; *cholecystokinin; *cholecystokinin receptors; *ligands; *luciferase; *proteins; *G protein-coupled cholecystokinin receptors --agonist-dependent oligomeric complex dissociation; *G protein-coupled cholecystokinin receptors --analysis; *G protein-coupled cholecystokinin receptors --functions; *G protein-coupled cholecystokinin receptors --structures; *G proteins; *bioluminescence resonance energy transfer technique --applications; *bioluminescence resonance energy transfer technique --molecular method; *bioluminescence resonance energy transfer technique --Molecular Biology Techniques and Chemical Characterization; *cell cultures --culture method; *cell cultures --Histological/Cytological and Culture Techniques; *co-immunoprecipitation --precipitation; *co-immunoprecipitation --precipitation method; *fluorescence assays --analytical method; *fluorescence assays --molecular method; *transfection --expression/vector techniques; *transfection --genetic method; *Spex fluorolog spectrofluorometer --laboratory equipment; *Spex fluorolog spectrofluorometer --Spex Industries; *Western blotting --analytical method; *Western blotting --labeling; *signaling

2001

Acquisition of a Phase-Modulated Fluorimeter for Materials Research and Education

Schwartz, Benjamin J

University of California-Los Angeles, Department of Chemistry and Biochemistry , 2001

This award from the Instrumentation for Materials Research program will support the University of California Los Angeles (UCLA) with the acquisition of a Fluorolog 3-22 Lifetime System from ISA/Jobin Yvon. The instrument consists of single excitation and double collection monochromators, with fully automated entrance and exit slit control. The instrument will have an immediate impact on the research projects of four research groups at UCLA. The first group will investigate interactions between chains of semiconducting polymers and the dynamics of energy flow in aligned conjugated polymer/mesoporous silica hybrid materials. The work should provide a new understanding of these important materials that will enhance the possibility of application in organic-based displays or photovoltaic devices. The second group will study the properties of newly synthesized molecular compasses and gyroscopes, as well as explore the effects of aromatic ring rotation in conjugated molecules. The information learned from these experiments will have direct application in the production of new organic materials that should have switching properties that are faster than the fastest known ferroelectric liquid crystals. The third group will explore the synthesis and properties of novel heteroacenes, which also will have unique applications in electroluminescent and photovoltaic devices. Finally, the fourth group will use time-resolved resonance energy transfer to study the spatial distributions of molecules deliberately placed in mesostructured silicas, and time-resolved fluorescence depolarization as an in situ probe of the formation dynamics of mesostructured silica films. The information gained in these experiments will improve our understanding of how the formation of mesostructured materials can be controlled and directed. The simplicity of the instrument will ensure routine usage and great productivity, especially in an undergraduate educational environment. Undergraduate students in the physical chemistry and analytical laboratory classes will be able to routinely measure the fluorescence lifetimes of dyes and other molecules, allowing them to explore the dynamics of intramolecular electron transfer and other important photochemical reactions. This award from the Instrumentation for Materials Research program will support the University of California Los Angeles (UCLA) with the acquisition of a Fluorolog 3-22 Lifetime System from ISA/Jobin Yvon. The instrument consists of single excitation and double collection monochromators, with fully automated entrance and exit slit control. The instrument will have an immediate impact on the research projects of four research groups at UCLA. The information gained in these experiments will improve our understanding of how the formation of mesostructured materials can be controlled and directed. The simplicity of the instrument will ensure routine usage and great productivity, especially in an undergraduate educational environment. Undergraduate students in the physical chemistry and analytical laboratory classes will be able to routinely measure the fluorescence lifetimes of dyes and other molecules, allowing them to explore the dynamics of intramolecular electron transfer and other important photochemical reactions.

2001

Accessory factors facilitate the binding of glucocorticoid receptor to the phosphoenolpyruvate carboxykinase gene promoter.

Granner, Daryl K.; Beechem, Joseph M.; Stafford, John M.; Wilkinson, John C.

Journal of Biological Chemistry, VOL. 276, NO. 43, October 26, 2001, PP. 39885-39891

Glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene requires a glucocorticoid response unit (GRU) comprised of two non-consensus glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least three accessory factor elements (gAF1-3). DNA-binding accessory proteins are commonly required for the regulation of genes whose products play an important role in metabolism, development, and a variety of defense responses, but little is known about why they are necessary. Quantitative, real time homogenous assays of cooperative protein-DNA interactions in complex media (e.g. nuclear extracts) have not previously been reported. Here we perform quantitative, real time equilibrium and stopped-flow fluorescence anisotropy measurements of protein-DNA interactions in nuclear extracts to demonstrate that GR binds to the GR1-GR2 elements poorly as compared with a palindromic or consensus glucocorticoid response element (GRE). Inclusion of either the gAF1 or gAF2 element with GR1-GR2, however, creates a high affinity binding environment for GR. GR can undergo multiple rounds of binding and dissociation to the palindromic GRE in less than 100 ms at nanomolar concentrations. The dissociation rate of GR is differentially slowed by the gAF1 or gAF2 elements that bind two functionally distinct accessory factors, COUP-TF/HNF4 and HNF3, respectively.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *H4IIE cell line (Muridae) --murine hepatoma cells; *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates; *accessory factors; *glucocorticoid receptor 1 GR1 --binding; *glucocorticoid receptor 2 GR2 --binding; *glucocorticoid response element GRE ; *glucocorticoid response unit GRU ; *gAF1; *gAF2; *gAF3; *phosphoenolpyruvate carboxykinase gene promoter; *COUP-TF/HNF3; *COUP-TF/HNF4; *quantitative real time homogenous assay --analytical method; *quantitative real time homogenous assay --Molecular Biology Techniques and Chemical Characterization; *stopped-flow fluorescence anisotropy --analytical method; *stopped-flow fluorescence anisotropy --Molecular Biology Techniques and Chemical Characterization; *Biologic SFM4 stopped-flow unit --laboratory equipment; *Biologic SFM4 stopped-flow unit --Molecular Kinetics; *SPEX 1681 fluorolog spectrofluorimeter --laboratory equipment; *SPEX 1681 fluorolog spectrofluorimeter --SPEX; *protein-DNA interaction