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

掲載年

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掲載誌

概要

2002

UV laser induced fluorescence spectroscopic studies and trace detection of dissolved plastics (bisphenol-A) and organic compounds in water

Sivaprakasam, Vasanthi, Major Professor: Dennis K. Killinger

VOL. 63-05B, 2002, PP. 2446

Two different Laser Induced Fluorescence (LIF) systems were developed and used to measure the fluorescence spectra of trace organic compounds and plastic residue leached into water. The first LIF system, a wavelength tunable UV laboratory system, was used for spectroscopic surveys and consisted of a nitrogen pumped, frequency-doubled, tunable (220 nm−280 nm) UV laser system. The collected fluorescence light (300 nm to 700 nm) was analyzed by a spectrograph and cooled CCD detector array. The system was used to obtain calibration fluorescence spectra and Excitation-Emission Matrix (EEM) spectra of Bisphenol-A and various water samples containing dissolved organics in the water. .A second LIF system, a portable fixed UV wavelength LIF system, was developed for shipborne measurements in detecting artificial and natural trace organic substances in seawater. The excitation laser was a fixed wavelength microchip laser at 266 nm and the collected fluorescence light was analyzed by optical interference filters ranging from 239 nm to 685 nm and a PMT detector. Studies were made to optimize the excitation source, collection optics, and detection process. Detailed fluorescence emission spectra due to the plastic bisphenol-A and the fluorescence standard quinine sulfate were made including lifetime, bleaching, and polarization measurements. .The signal to noise ratio of the portable and the Laboratory LIF systems were measured and compared to a commercial laboratory Spectrofluorometer ( Fluoro Max ) and a portable fluorescence system (SAFire) both using Xenon light sources for excitation. It was found that the detection limit of the portable LIF system was a factor of 500 better than the commercial SAFire in terms of quinine sulfate and a factor of 50 higher than the Fluoro Max .The portable system was tested on a 2 day cruise into the Gulf of Mexico to determine the instrument's sensitivity in a variety of water masses. The portable LIF system was able to measure dissolved organic compounds with a fluorescence emission near 450 nm in clean “blue” Gulf water with a sensitivity about two orders of magnitude greater than other previous measurements using the SAFire instrument.

DESCRIPTOR(S)- Physics, Optics

2002

Three topologically equivalent core residues affect the transition state ensemble in a protein folding reaction.

Jennings, Patricia A.; Heidary, David K.

Journal of Molecular Biology. VOL. 316, NO. 3, 22 February, 2002, PP. 789-798

The single domain protein, interleukin-1beta, is representative of a distinct class of proteins characterized by their beta-trefoil topology. Each subdomain of this structural class is composed of a beta beta beta loop beta (betabetabetaLbeta) motif comprised of apprx50 residues and gives the protein a pseudo-3-fold axis of symmetry. A common feature of proteins in this topological family appears to be that they are slow folders, which reach the native state on the order of tens to 100s of seconds. Sequence analysis of interleukin-1beta indicates that three phenylalanine residues located at positions 42, 101, and 146 are well conserved, separated by apprx50 residues in the primary sequence, located in similar positions in the pseudo-symmetric units of the trefoil, and are juxtaposed to one another in conformational space. These residues surround the hydrophobic cavity and "pin" the hairpin triplet cap to the core beta-barrel. To determine if cap-barrel interactions are involved in maintaining the structural stability and cooperativity or in controlling the slow formation of the native state, we performed a series of mutational studies. The results indicate that interleukin-1beta tolerates large increases in side-chain volume at these three topologically conserved sites with little effect on stability, while the kinetics show significant differences in both the unfolding and refolding rates. Taken together, our results indicate that these conserved core residues are essential contacts in the transition-state ensemble for folding.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *interleukin-1-beta --beta-trefoil topology; *phenylalanine --topologically equivalent core residues; *equilibrium titration --biochemical method; *equilibrium titration --Molecular Biology Techniques and Chemical Characterization; *manual-mixing circular dichroism kinetics --analytical method; *manual-mixing circular dichroism kinetics --Spectrum Analysis Techniques; *manual-mixing fluorescence kinetics --analytical method; *manual-mixing fluorescence kinetics --Spectrum Analysis Techniques; *mutational studies --analytical method; *mutational studies --Molecular Biology Techniques and Chemical Characterization; *sequence analysis --analytical method; *sequence analysis --Molecular Biology Techniques and Chemical Characterization; *site-directed mutagenesis --analytical method; *site-directed mutagenesis --mutagenesis/deletion; *site-directed mutagenesis --protein engineering; *stopped-flow fluorescence --analytical method; *stopped-flow fluorescence --Spectrum Analysis Techniques; *Applied Photophysics SX.17MV unit --laboratory equipment; *Applied Photophysics SX.17MV unit --Applied Photophysics; *DNA sequencing --molecular genetic method; *DNA sequencing --recombinant DNA technology; *DNA sequencing --sequencing techniques; * Fluoromax II spectrophotometer --laboratory equipment; * Fluoromax II spectrophotometer --Spex; * Fluoromax-2 spectrofluorimeter --laboratory equipment; * Fluoromax-2 spectrofluorimeter --Spex; *60 DS Spectropolarimeter --laboratory equipment; *60 DS Spectropolarimeter --Aviv Instruments; *protein folding reaction; *transition state ensemble

2002

The fluorescence spectrum of the introduced tryptophans in the alpha3 (betaF155W) 3gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites.

Allison, William S.; Dong, Ken; Ren, Huimiao

Journal of Biological Chemistry, VOL. 277, NO. 11, March 15, 2002, PP. 9540-9547

It has been reported that shifts in the fluorescence emission spectrum of the introduced tryptophans in the betaF155W mutant of Escherichia coli F1 (bovine heart mitochondria F1 residue number) can quantitatively distinguish between the number of catalytic sites occupied with ADP and ATP during steady-state ATP hydrolysis (Weber, J., Bowman, C., and Senior, A. E. (1996) J. Biol. Chem. 271, 18711-18718). In contrast, addition of MgADP, Mg-5'-adenylyl beta,gamma-imidophosphate (MgAMP-PNP), and MgATP in 1:1 ratios to the alpha3(betaF155W)3gamma subcomplex of thermophilic Bacillus PS3 F1 (TF1) induced nearly identical blue shifts in the fluorescence emission maximum that was accompanied by quenching. Addition of 2 mM MgADP induced a slightly greater blue shift and a slight increase in intensity over those observed with 1:1 MgADP. However, addition of 2 mM MgAMP-PNP or MgATP induced a much greater blue shift and substantially enhanced fluorescence intensity over those observed in the presence of stoichiometric MgADP or MgAMP-PNP. It is clear from these results that the fluorescence spectrum of the introduced tryptophans in the betaF155W mutant of TF1 does not respond in regular increments at any wavelength as catalytic sites are filled with nucleotides. The fluorescence spectrum observed after entrapping MgADP-fluoroaluminate complexes in two catalytic sites of the betaF155W subcomplex indicates that the fluorescence emission spectrum of the enzyme is maximally perturbed when nucleotides are bound to two catalytic sites. This finding is consistent with accumulating evidence suggesting that only two beta subunits in the alpha3beta3gamma subcomplex of TF1 can simultaneously exist in the completely closed conformation.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Bacillus sp. (Endospore-forming Gram-Positives) --strain-PS3; *Bacillus sp. (Endospore-forming Gram-Positives) --thermophilic; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *F1-ATPase --alpha3(betaF155W)3gamma subcomplex; *F1-ATPase --analysis; *F1-ATPase --bound nucleoside diphosphates; *F1-ATPase --bound nucleoside triphosphates; *F1-ATPase --catalytic activity; *F1-ATPase --tryptophan introduction; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *Spex Fluoromax-2 spectrofluorometer --laboratory equipment

2002

The activation of RalGDS can be achieved independently of its Ras binding domain. Implications of an activation mechanism in Ras effector specificity and signal distribution.

Herrmann, Christian; Herter, Peter; Kiel, Christina; Linnemann, Thomas

Journal of Biological Chemistry, VOL. 277, NO. 10, March 8, 2002, PP. 7831-7837

Small GTPases of the Ras family are major players of signal transduction in eukaryotic cells. They receive signals from a number of receptors and transmit them to a variety of effectors. The distribution of signals to different effector molecules allows for the generation of opposing effects like proliferation and differentiation. To understand the specificity of Ras signaling, we investigated the activation of RalGDS, one of the Ras effector proteins with guanine-nucleotide exchange factor activity for Ral. We determined the GTP level on RalA and showed that the highly conserved Ras binding domain (RBD) of RalGDS, which mediates association with Ras, is important but not sufficient to explain the stimulation of the exchange factor. Although a point mutation in the RBD of RalGDS, which abrogates binding to Ras, renders RalGDS independent to activated Ras, an artificially membrane-targeted version of RalGDS lacking its RBD could still be activated by Ras. The switch II region of Ras is involved in the activation, because the mutant Y64W in this region is impaired in the RalGDS activation. Furthermore, it is shown that Rap1, which was originally identified as a Ras antagonist, can block Ras-mediated RalGDS signaling only when RalGDS contains an intact RBD. In addition, kinetic studies of the complex formation between RalGDS-RBD and Ras suggest that the fast association between RalGDS and Ras, which is analogous to the Ras/Raf case, achieves signaling specificity. Conversely, the RascntdotRalGDS complex has a short lifetime of 0.1 s and Rap1 forms a long-lived complex with RalGDS, possibly explaining its antagonistic effect on Ras.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *COS7 cell line (Cercopithecidae) --African green monkey kidney cells; *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Primates; *Nonhuman Vertebrates; *Primates; *Vertebrates; *Ral; *RalA; *RalGDS --activation; *RalGDS --point mutation; *RalGDS --Ras binding domain; *Rap1; *Ras; *Ras effector proteins; *Ras GTPases; *cell culture --culture method; *cell culture --Histological/Cytological and Culture Techniques; *immunofluorescence --analytical method; *immunofluorescence --Immunologic Techniques; *in vitro assay --bioassay method; *in vitro assay --Bioassays/Physiological Analysis; *size exclusion chromatography --liquid chromatography; *size exclusion chromatography --purification method; *transfection assay --bioassay method; *transfection assay --Bioassays/Physiological Analysis; * Fluoromax spectrofluorometer --laboratory equipment; * Fluoromax spectrofluorometer --Spex; *Superdex 75 column --laboratory equipment; *Superdex 75 column --Amersham Biosciences Inc.; *Western blot --analytical method; *Western blot --gene mapping; *Western blot --labeling; *Zeiss confocal microscope --laboratory equipment; *Zeiss confocal microscope --Zeiss; *signal transduction

2002

Subunit exchange and the role of dimer flexibility in DNA binding by the Fis protein.

Johnson, Reid C.; Merickel, Stacy K.; Sanders, Erin R.; Vazquez-Ibar, Jose Luis

Biochemistry, VOL. 41, NO. 18, May 7, 2002, PP. 5788-5798

Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Fis protein --bacterial DNA binding protein; *Fis-DNA complex; *fluorescence spectroscopy --Spectrum Analysis Techniques; *FRET spectroscopy fluorescence resonance energy transfer spectroscopy --Spectrum Analysis Techniques; *SPEX Fluoromax-3 spectrofluorometer --Jobin-Yvon Horiba SPEX; *SPEX Fluoromax-3 spectrofluorometer --Spectrum Analysis Techniques; *DNA bending; *DNA binding; *DNA binding affinities; *DNA binding dimer flexibility; *DNA binding subunit exchange; *Fis protein-DNA binding --role of dimer flexibility; *Fis protein-DNA binding --subunit exchange; *X-ray crystal structures

2002

Studies of potential cerebrospinal fluid molecular markers for Alzheimer's disease.

Lee, Kelvin H.; Choe, Leila H.; Dutt, Michael J.; Relkin, Norman

Electrophoresis, VOL. 23, NO. 14, July, 2002, PP. 2247-2251

There is a need for a reliable, molecular-based ante mortem diagnostic test for Alzheimer's disease (AD). In this study, we examined the use of two-dimensional protein electrophoresis for generating molecular barcodes which may be useful for the clinical differentiation of AD patients from normals. We compared cerebrospinal fluid samples taken from AD patients with confirmed post mortem pathology to comparable specimens from normal volunteers. Using canonical correlation analysis, a panel of nine molecular markers were identified which segregated diseased cases from normal controls. Using the scaled volume image analysis variable, a principal factor analysis was also used to distinguish normal from AD spinal fluid, based on molecular markers identified using a heuristic clustering algorithm. The use of panels of molecular markers derived from proteomic analysis may offer the best prospect for developing molecular diagnostic tests for complex neurodegenerative disorders such as AD.

DESCRIPTOR(S)- *Clinical Chemistry (Allied Medical Sciences); *Methods and Techniques; *Neurology (Human Medicine, Medical Sciences); *human (Hominidae) --patient; *human (Hominidae) --volunteer; *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *cerebrospinal fluid --nervous system; *Alzheimer's disease --behavioral and mental disorders; *Alzheimer's disease --nervous system disease; *canonical correlation analysis --evaluation method; *canonical correlation analysis --Molecular Biology Techniques and Chemical Characterization; *isoelectric focusing gels --laboratory equipment; *isoelectric focusing gels --Amersham Biosciences; *molecular marker identification --identification method; *molecular marker identification --Molecular Biology Techniques and Chemical Characterization; *two-dimensional protein electrophoresis --separation method; *two-dimensional protein electrophoresis --Electrophoretic Techniques; *vertical gradient slab gels --laboratory equipment; *vertical gradient slab gels --Bio-Rad; *Bio-Rad GS525 imaging system --laboratory equipment; *Bio-Rad GS525 imaging system --Bio-Rad; *ELISA kit --laboratory kit; *ELISA kit --Biosource International; *ISA SPEX FluoroMax 2 fluorimeter --laboratory equipment; *MicroMax plate reader --laboratory equipment; *Multiphor Instrument --laboratory equipment; *Multiphor Instrument --Amersham Biosciences; *SuperSignal West Dura kit --laboratory kit; *SuperSignal West Dura kit --Pierce; *Alzheimer Disease (MeSH)

2002

Structure and calcium-binding studies of a recoverin mutant (E85Q) in an allosteric intermediate state.

Ames, James B.; Hamasaki, Nobuko; Molchanova, Tatiana

Biochemistry, VOL. 41, NO. 18, May 7, 2002, PP. 5776-5787

Recoverin, a member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl or related fatty acyl group covalently attached to the N-terminus of recoverin facilitates the binding of recoverin to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Previous structural studies revealed that the myristoyl group of recoverin is sequestered inside the protein core in the absence of calcium. The cooperative binding of two calcium ions to the second and third EF-hands (EF-2 and EF-3) of recoverin leads to the extrusion of the fatty acid. Here we present nuclear magnetic resonance (NMR), fluorescence, and calcium-binding studies of a myristoylated recoverin mutant (myr-E85Q) designed to abolish high-affinity calcium binding to EF-2 and thereby trap the myristoylated protein with calcium bound solely to EF-3. Equilibrium calcium-binding studies confirm that only one Ca2+ binds to myr-E85Q under the conditions of this study with a dissociation constant of 100 muM. Fluorescence and NMR spectra of the Ca2+-free myr-E85Q are identical to those of Ca2+-free wild type, indicating that the E85Q mutation does not alter the stability and structure of the Ca2+-free protein. In contrast, the fluorescence and NMR spectra of half-saturated myr-E85Q (one bound Ca2+) look different from those of Ca2+-saturated wild type (two bound Ca2+), suggesting that half-saturated myr-E85Q may represent a structural intermediate. We report here the three-dimensional structure of Ca2+-bound myr-E85Q as determined by NMR spectroscopy. The N-terminal myristoyl group of Ca2+-bound myr-E85Q is sequestered within a hydrophobic cavity lined by many aromatic residues (F23, W31, Y53, F56, F83, and Y86) resembling that of Ca2+-free recoverin. The structure of Ca2+-bound myr-E85Q in the N-terminal region (residues 2-90) is similar to that of Ca2+-free recoverin, whereas the C-terminal region (residues 100-202) is more similar to that of Ca2+-bound wild type. Hence, the structure of Ca2+-bound myr-E85Q represents a hybrid between the structures of recoverin with zero and two Ca2+ bound. The binding of Ca2+ to EF-3 leads to local structural changes within the EF-hand that alter the domain interface and cause a 45degree swiveling of the N- and C-terminal domains, resulting in a partial unclamping of the myristoyl group. We propose that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *myristoylated recoverin mutant myr-E85Q --calcium-binding studies; *myristoylated recoverin mutant myr-E85Q --structure; *fluorescence spectroscopy --Spectrum Analysis Techniques; *site-directed mutagenesis --mutagenesis/deletion; *site-directed mutagenesis --protein engineering; *Bruker DRX-500 spectrometer --Bruker; *Bruker DRX-500 spectrometer --Spectrum Analysis Techniques; *Bruker DRX-600 spectrometer --Bruker; *Bruker DRX-600 spectrometer --Spectrum Analysis Techniques; * FluoroMax-2 fluorometer --Jobin YVON-SPEX Instruments; * FluoroMax-2 fluorometer --Spectrum Analysis Techniques; *NMR spectroscopy --Spectrum Analysis Techniques; *allosteric intermediate state; *allosteric transition intermediate species; *calcium-myristoyl switch

2002

Structural features of model glycopeptides in solution and in membrane phase: A spectroscopic and molecular mechanics investigation.

Pispisa, B.; Carafa, M.; Maccaroni, E.; Palleschi, A.; Stella, L.; Straccamore, M. E.; Venanzi, M.; Zanotti, G.

Biopolymers, VOL. 64, NO. 1, June, 2002, PP. 44-56

Model glycopeptides of the general formula Boc-Ala-Thr(G-D)-A1-A2-Leu-Leu-Lys(N)-Ala-OMe, where D = dansyl (dimethyl aminonaphthalenesulphonyl), G = glucosyl and N = naphthyl, while A1-A2 = Ala-Leu or Aib-Aib, and denoted as D-G-Ala-N and D-G-Aib-N, respectively, were used to investigate glycoprotein-membrane interactions. They carry two fluorophores (D and N), covalently linked to the glucose ring and the lysine side chain, respectively, while the threonine side chain is O-glycosylated. CD spectra in different solvent media suggest that both glycopeptides attain an ordered structure, possibly a helix-like conformation. By combining FRET (fluorescence resonance energy transfer) experiments with molecular mechanics data, the most probable structures of both glycopeptides were built up, starting from both a right-handed (rh) alpha- and 310-helix. They were found to populate an alpha-helical conformation, a result further confirmed by the very good agreement between theoretical and experimental quenching efficiency only observed when the backbone chain was in alpha-helix. The association of D-G-Ala-N with model membranes (liposomes) was studied by CD, fluorescence decay, fluorescence anisotropy, and collisional quenching experiments. The binding does not alter the structural features of the peptide because the CD spectral patterns are unaffected by the association. The peptide orientation inside the phospholipidic bilayer is guided by the polar glucose molecule lying in the water phase. The insertion of the hydrophobic backbone chain into the membrane, seeing the probes only partially accessible from the external solution, is characterized by a significant degree of heterogeneity, an increase in vesicles size, and a relevant stabilizing effect on the membrane itself against rupture by methanol.

DESCRIPTOR(S)- *Chemistry; *Methods and Techniques; *methanol; *model glycopeptides --structural membrane phase features; *model glycopeptides --structural solution features; *fluorescence resonance energy transfer --detection method; *CD spectra --Spectrum Analysis Techniques; *JASCO J-600 CD spectra --Jasco; *JASCO J-600 CD spectra --Spectrum Analysis Techniques; *NMR spectroscopy --Spectrum Analysis Techniques; *SPEX Fluoromax fluorimeter --Molecular Biology Techniques and Chemical Characterization; *SPEX Fluoromax fluorimeter --SPEX; *collisional fluorescence quenching; *liposomes; *model membraned; *molecular mechanics calculations; *steady-state fluorescence; *time-resolved fluorescence

2002

Spectrofluorimetric determination of hydrogen peroxide scavenging activity.

Pazdzioch-Czochra, Marzanna; Widenska, Anna

Analytica Chimica Acta, VOL. 452, NO. 2, 11 February, 2002, PP. 177-184

Homovanillic acid (HVA) is widely used for the detection and imaging of oxidative enzymes-peroxidase, glucose oxidase and xanthine oxidase, but antioxidant activity has not been determined so far with the use of HVA. We have developed a simple, sensitive and in-field spectrofluorimetric method for the determination of hydrogen peroxide (H2O2) scavenging activity. The assay is based on the oxidation of HVA to its fluorescent biphenyl dimer in the presence of H2O2 and peroxidase. The presence of substances with H2O2 scavenging activity prevents the oxidation of HVA by removing H2O2. The decrease in fluorescence intensity is proportional to the antioxidative (H2O2 scavenging) activity. The method was evaluated using Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), BHA (3-t-butyl-4-hydroxyanisole) and ferulic, vanillic, caffeic, chlorogenic, protocatechuic and oxalic acids. Additionally, tea and herb infusions known for their antioxidant properties were evaluated.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *glucose oxidase; *homovanillic acid --Sigma; *hydrogen peroxide --free radical; *hydrogen peroxide --Merck; *phenolic compounds; *xanthine oxidase; *spectrofluorimetry --detection method; *spectrofluorimetry --fluorometry; * FluoroMax-2 spectrofluorimeter --laboratory equipment; *HPLC --purification method; *HPLC --Extraction; *HPLC --Isolation; *HPLC --Purification and Separation Techniques; *SDS-PAGE --analytical method; *SDS-PAGE --Electrophoretic Techniques

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

Probing for preferential interactions among sphingolipids in bilayer vesicles using the glycolipid transfer protein.

Mattjus, Peter; Brown, Rhoderick E.; Kline, Adam; Molotkovsky, Julian G.; Pike, Helen M.

Biochemistry, VOL. 41, NO. 1, January 8, 2002, PP. 266-273

We have investigated the intervesicular transfer of galactosylceramide between unilamellar bilayer vesicles composed of differing sphingomyelin and phosphatidylcholine molar ratios. To monitor glycolipid transfer from donor to acceptor vesicles, we used a fluorescence resonance energy transfer assay involving anthrylvinyl-labeled galactosylceramide (AV-GalCer) and perylenoyl-labeled triglyceride. The transfer was mediated by glycolipid transfer protein (GLTP), purified from bovine brain and specific for glycolipids. The initial transfer rate and the total accessible pool of glycolipid in the donor vesicles were both measured. An increase in the sphingomyelin content of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles decreased the transfer rate in a nonlinear fashion. Decreased transfer rates were clearly evident at sphingomyelin mole fractions of 0.22 or higher. The pool of AV-GalCer available for GLTP-mediated transfer also was smaller in vesicles containing high sphingomyelin content. In contrast, AVGalCer was more readily transferred from vesicles composed of POPC and different disaturated phosphatidylcholines. Our results show that GLTP acts as a sensitive probe for detecting interactions of glycosphingolipids with neighboring lipids and that the lateral mixing of glycolipids is probably affected by the matrix lipid composition. The compositionally driven changes in lipid interactions, sensed by GLTP, occur in membranes that are either macroscopically fluid-phase or gel/fluid-phase mixtures. Gaining insights into how changes in membrane sphingolipid composition alter accessibility to soluble proteins with affinity for membrane glycolipids is likely to help increase our understanding of how sphingolipid-enriched microdomains (i.e., "rafts" and caveolae) are formed and maintained in cells.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *bovine (Bovidae); *Animals; *Artiodactyls; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Vertebrates; *brain --nervous system; *membranes --fluid-phase; *membranes --gel/fluid-phase mixture; *unilamellar bilayer vesicles; *anthrylvinyl-labeled galactosylceramide AV-GalCer --fluorescent probe; *anthrylvinyl-labeled galactosylceramide AV-GalCer --intervesicular transfer; *disaturated phosphatidylcholines disaturated PCs --analysis; *glycolipid transfer protein GLTP --analysis; *perylenoyl-labeled triglyceride Per-TG --fluorescent probe; *sphingolipids --analysis; *sphingomyelin SPM --analysis; *sphingomyelin SPM --Avanti Polar Lipids; *1-palmitoyl-2-oleoyl phosphatidylcholine POPC --analysis; *1-palmitoyl-2-oleoyl phosphatidylcholine POPC --Avanti Polar Lipids; *fluorescence resonance energy transfer assay FRET assay --monitoring method; *fluorescence resonance energy transfer assay FRET assay --Bioassays/Physiological Analysis; *Neslab RTE-111 circulating water bath --laboratory equipment; *Neslab RTE-111 circulating water bath --Neslab Instruments; *SPEX Fluoromax instrument --laboratory equipment; *SPEX Fluoromax instrument --Inc.; *SPEX Fluoromax instrument --Instruments SA; *molar ratios; *preferential interactions

2002

Mutagenic scan of the H-N-H motif of colicin E9: Implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases.

James, Richard; Georgiou, Theonie; Kleanthous, Colin; Moore, Geoffrey R.; Pommer, Ansgar J.; Walker, Daniel; Walker, David C.

Nucleic Acids Research, VOL. 30, NO. 14, July 15, 2002, PP. 3225-3234

Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H-N-H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H-N-H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H-N-H motif is the site of DNA binding and that Mg2+-dependent cleavage of double-stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation-dependent catalytic mechanisms. The roles of individual amino acids within the H-N-H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase-activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H-N-H DNases.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *bacteria (Bacteria); *Escherichia coli (Enterobacteriaceae) --strain-BL21 DE3; *Escherichia coli (Enterobacteriaceae) --strain-ER2566; *Bacteria; *Eubacteria; *Microorganisms; *apoptotic endonucleases; *caspase-activated DNases; *chromosomal DNA --random degradation; *colicin E9 --toxin; *colicin E9 --H-N-H motif; *colicins; *homing enzymes; *metal ion --binding; *pET vectors --Novagen; *DNA --hydrolysis; *mutagenic scan --analytical method; *mutagenic scan --Molecular Biology Techniques and Chemical Characterization; *Philips PU 8730 spectrophotometer --laboratory equipment; *Philips PU 8730 spectrophotometer --Philips; *QIAEX-II gel extraction kit --laboratory kit; *QIAEX-II gel extraction kit --Qiagen; *Spex Fluoromax 3 spectrofluorimeter --laboratory equipment; *Spex Fluoromax 3 spectrofluorimeter --Spex; *Storm 840 phosphoimager --laboratory equipment; *Storm 840 phosphoimager --Molecular Dynamics; *mechanistic enzymology
BIOLOGICAL TAXONOMIC DESCRIPTOR(S)- Bacteria --Microorganisms; Enterobacteriaceae --Bacteria; Enterobacteriaceae --Eubacteria; Enterobacteriaceae --Facultatively Anaerobic Gram-Negative Rods; Enterobacteriaceae --Microorganisms

2002

Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose.

Tjerneld, Folke; Borjesson, Johan; Eriksson, Torny

Enzyme and Microbial Technology, VOL. 31, NO. 3, 2 August, 2002, PP. 353-364

Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore, experiments were designed to explore mechanisms of surfactant effects. A number of surfactants were screened for their ability to improve enzymatic hydrolysis of steam-pretreated spruce (SPS). Non-ionic surfactants were found to be the most effective. Studies of adsorption of the dominating cellulase of Trichoderma reesei, Cel7A (CBHI), during hydrolysis showed that the anionic and non-ionic surfactants reduced enzyme adsorption to the lignocellulose substrate. The approximate reduction of enzyme adsorption was from 90% adsorbed enzyme to 80% with surfactant addition. Cellulase stability in the presence of surfactants was studied by activity and fluorescence measurements. Surfactants were shown to have only a weak effect on cellulase temperature stability. Our conclusions from studies of lignocellulose and delignified substrates are that the improved conversion of lignocellulose with surfactant can be explained by the reduction of the unproductive enzyme adsorption to the lignin part of the substrate. This is due to hydrophobic interaction of surfactant with lignin on the lignocellulose surface, which releases unspecifically bound enzyme. A new approach with mixed charged and non-ionic surfactants has been introduced to further improve the positive effect of the surfactant addition.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Metabolism; *Methods and Techniques; *Trichoderma reesei (Fungi Imperfecti or Deuteromycetes); *Fungi; *Microorganisms; *Nonvascular Plants; *Plants; *cellulose; *ethanol; *lignin; *lignocellulose; *surfactant; *Cel7A --cellulase; *Cel7A --temperature stability; *anion exchange chromatography --column chromatography; *anion exchange chromatography --detection method; *anion exchange chromatography --Chromatographic Techniques; *enzymatic hydrolysis --enzymatic method; *enzymatic hydrolysis --Molecular Biology Techniques and Chemical Characterization; *liquid scintillation --detection method; *liquid scintillation --Molecular Biology Techniques and Chemical Characterization; *syringe filter --laboratory equipment; *syringe filter --Millipore; *Beckman liquid scintillator LS 1801 --laboratory equipment; *Beckman liquid scintillator LS 1801 --Beckman; *CarboPac PA100 analytical column --laboratory equipment; *CarboPac PA100 analytical column --Dionex; *DX 500 High Performance Anion Exchange Chromatography system with Pulsed Amperometric Detection --laboratory equipment; *DX 500 High Performance Anion Exchange Chromatography system with Pulsed Amperometric Detection --Dionex; * Fluoromax-2 spectrofluorometer --laboratory equipment; * Fluoromax-2 spectrofluorometer --Instruments S.A. Inc.; *steam-pretreated spruce

2002

Helicase from hepatitis C virus, energetics of DNA binding.

Patel, Smita S.; Levin, Mikhail K.

Journal of Biological Chemistry, VOL. 277, NO. 33, August 16, 2002, PP. 29377-29385

The ability of a helicase to bind single-stranded nucleic acid is critical for nucleic acid unwinding. The helicase from the hepatitis C virus, NS3 protein, binds to the 3'-DNA or the RNA strand during unwinding. As a step to understand the mechanism of unwinding, DNA binding properties of the helicase domain of NS3 (NS3h) were investigated by fluorimetric binding equilibrium titrations. The global analysis of the binding data by a combinatorial approach was done using MATLAB. NS3h interactions with single-stranded DNA (ssDNA) are 300-1000-fold tighter relative to duplex DNA. The NS3h protein binds to ssDNA less than 15 nt in length with a stoichiometry of one protein per DNA. The minimal ssDNA binding site of NS3h helicase was determined to be 8 nucleotides with the microscopic Kd of 2-4 nM or an observed free energy of -50 kJ/mol. These NS3h-DNA interactions are highly sensitive to salt, and the Kd increases 4 times when the NaCl concentration is doubled. Multiple HCV helicase proteins bind to ssDNA >5 nucleotides in length, with an apparent occluded site of 8-11 nucleotides. The DNA binding data indicate that the interactions of multiple NS3h protein molecules with long ssDNA are both noncooperative and sequence-independent. We discuss the DNA binding properties of HCV helicase in relation to other superfamily 1 and 2 helicases. These studies provide the basis to investigate the DNA binding interactions with the unwinding substrate and their modulation by the ATPase activity of HCV helicase.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *hepatitis C virus (Flaviviridae); *Animal Viruses; *Microorganisms; *Viruses; *sodium chloride; *superfamily 1 helicase; *superfamily 2 helicase; *DNA --duplex; *DNA --single-stranded; *NS3 protein --helicase domains; *NS3 protein --ATPase activity; *RNA; *combinatorial global binding data analysis --analytical method; *combinatorial global binding data analysis --computer method; *combinatorial global binding data analysis --Mathematical and Computer Techniques; *fluorimetric binding equilibrium titration --analytical method; *fluorimetric binding equilibrium titration --Molecular Biology Techniques and Chemical Characterization; * FluoroMax-2 spectrofluorimeter --laboratory equipment; * FluoroMax-2 spectrofluorimeter --Spex Instruments S.A. Inc.; *MATLAB --computer software; *Oligo Analyzer --computer software; *PhosphorImager --laboratory equipment; *PhosphorImager --Molecular Dynamics; *8452A Diode Array Spectrophotometer --laboratory equipment; *8452A Diode Array Spectrophotometer --Hewlett Packard; *nucleic acid unwinding; *DNA binding energetics

2002

Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe.

Jonas, Ana; Agree, Andrea K. Behling; McGuire, Kirsten Arnvig; Tian, Shao-Min; Tricerri, M. Alejandra

Biochimica et Biophysica Acta, VOL. 1594, NO. 2, 11 February, 2002, PP. 286-296

Apolipoprotein A-I (apoA-I) has important physiologic roles in reverse cholesterol transport, as a component of HDL; however, apoA-I also exists in lipid-poor or lipid-free forms that are key intermediates in HDL metabolism and acceptors of lipids from cells. The aim of this study was to examine the structure and stability of the central and C-terminal regions of lipid-free apoA-I. To this end, five Cys mutants of proapoA-I were constructed and expressed in Escherichia coli: V119C, A124C, A154C, A190C, and A232C. These mutants were specifically labeled with 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AC) and were examined by CD spectroscopy and a variety of fluorescence methods. The results showed that the introduction of Cys residues and their covalent labeling with AC did not affect the overall structure and stability of apoA-I. However, AC fluorescence properties revealed that different segments of the central and C-terminal half of apoA-I have distinct folding and stability properties. From fluorescence energy transfer data, average distances between the N-terminal region containing Trp residues and the various AC locations were obtained. The current results, together with previously published observations, led to the construction of a three-dimensional model for the folding of lipid-free apoA-I.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *human (Hominidae); *Escherichia coli (Enterobacteriaceae) --expression method; *Animals; *Bacteria; *Chordates; *Eubacteria; *Humans; *Mammals; *Microorganisms; *Primates; *Vertebrates; *apolipoprotein A-I --folding activity; *apolipoprotein A-I --stability; *apolipoprotein A-I --C-terminal half; *cholesterol --reverse transport; *cysteine; *lipid; *tryptophan; *A124C --cysteine mutant; *A154C --cysteine mutant; *A190C --cysteine mutant; *A232C --cysteine mutant; *HDL high density lipoprotein ; *V119C --cysteine mutant; *6-acryloyl-2-dimethylaminonaphthalene AC, acrylodan --fluorescent label; *6-acryloyl-2-dimethylaminonaphthalene AC, acrylodan --Molecular Probes; *cysteine mutant construction --synthetic method; *cysteine mutant construction --Synthetic Techniques; *cysteine mutant expression --biochemical method; *cysteine mutant expression --Molecular Biology Techniques and Chemical Characterization; *fluorometer --laboratory equipment; *fluorometer --ISS Inc.; *laser --laboratory equipment; *laser --Coherent; *site-directed mutagenesis --mutagenesis/deletion; *site-directed mutagenesis --protein engineering; *site-directed mutagenesis --synthetic method; *site-directed mutagenesis --Synthetic Techniques; *CD spectroscopy circular dichroism spectroscopy --visualization method; *CD spectroscopy circular dichroism spectroscopy --Spectrum Analysis Techniques; *ISA SPEX Fluoromax photon-counting spectrofluorometer --laboratory equipment; *J-720 spectropolarimeter --laboratory equipment; *KV 399 long pass filter --laboratory equipment; *KV 399 long pass filter --Schott; *Quickchange method --synthetic method; *Quickchange method --Synthetic Techniques

2002

Folding and oxidation of the antibody domain CH3.

Buchner, Johannes; Bell, Stefan; Marino, Gennaro; Mayer, Marcus; Ruoppolo, Margherita; Talamo, Fabio; Thies, Michael J. W.

Journal of Molecular Biology, VOL. 319, NO. 5, 21 June, 2002, PP. 1267-1277

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (CH3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced CH3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced CH3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded CH3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of CH3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Chemistry; *Immune System (Chemical Coordination and Homeostasis); *Methods and Techniques; *antibody domain C-H-3 --folding; *antibody domain C-H-3 --oxidation; *cysteine residues; *immunoglobulin; *circular dichroism spectroscopy --Spectrum Analysis Techniques; *electrospray ionization-mass spectrometry --mass spectrometry; *fluorescence spectroscopy --Spectrum Analysis Techniques; *matrix-assisted laser/desorption ionization-time-of-flight mass spectrometry --mass spectrometry; *Jasco J-715 spectropolarimeter --Jasco; *Jasco J-715 spectropolarimeter --Molecular Biology Techniques and Chemical Characterization; *Jasco J-715 spectropolarimeter --Spectrum Analysis Techniques; *Spex FluoroMax-2 fluorimeter --Molecular Biology Techniques and Chemical Characterization; *Spex FluoroMax-2 fluorimeter --Spectrum Analysis Techniques; *Voyager DE mass spectrometer --PerSeptive Biosystems; *Voyager DE mass spectrometer --Spectrum Analysis Techniques; *oxidation; *protein conformation; *protein stability

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 reactions

2002

Elucidation of the molecular mechanism during the early events in immunoglobulin light chain amyloid fibrillation. Evidence for an off-pathway oligomer at acidic pH.

Fink, Anthony L.; Doniach, Sebastian; Khurana, Ritu; Millett, Ian S.; Souillac, Pierre O.; Uversky, Vladimir N.

Journal of Biological Chemistry, VOL. 277, NO. 15, April 12, 2002, PP. 12666-12679

Light chain amyloidosis involves the systemic pathologic deposition of monoclonal light chain variable domains of immunoglobulins as insoluble fibrils. The variable domain LEN was obtained from a patient who had no overt amyloidosis; however, LEN forms fibrils in vitro, under mildly destabilizing conditions. The in vitro kinetics of fibrillation were investigated using a wide variety of probes. The rate of fibril formation was highly dependent on the initial protein concentration. In contrast to most amyloid systems, the kinetics became slower with increasing LEN concentrations. At high protein concentrations a significant lag in time was observed between the conformational changes and the formation of fibrils, consistent with the formation of soluble off-pathway oligomeric species and a branched pathway. The presence of off-pathway species was confirmed by small angle x-ray scattering. At low protein concentrations the structural rearrangements were concurrent with fibril formation, indicating the absence of formation of the off-pathway species. The data are consistent with a model for fibrillation in which a dimeric form of LEN (at high protein concentration) inhibits fibril formation by interaction with an intermediate on the fibrillation pathway and leads to formation of the off-pathway intermediate.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Chemistry; *Mathematical Biology (Computational Biology); *Methods and Techniques; *attenuated total reflectance-Fourier transform IR spectroscopy ATR-FTIR spectroscopy --Spectrum Analysis Techniques; *small-angle X-ray scattering --X-ray analysis; * FluoroMax-2 fluorescence spectrophotometer --Jobin Yvon-Spex; * FluoroMax-2 fluorescence spectrophotometer --Spectrum Analysis Techniques; *JEOL JEM-100B microscope --Histological/Cytological and Culture Techniques; *JEOL JEM-100B microscope --JEOL; *Nicolet 800 spectrophotometer --Nicolet; *Nicolet 800 spectrophotometer --Spectrum Analysis Techniques; *acidic pH; *fibril formation kinetics; *immunoglobulin light chain amyloid fibrillation --early event molecular mechanisms; *in vitro fibril formation procedure; *light chain amyloidosis; *off-pathway oligomer

2002

Effect of association state and conformational stability on the kinetics of immunoglobulin light chain amyloid fibril formation at physiological pH.

Fink, Anthony L.; Doniach, Sebastian; Khurana, Ritu; Millett, Ian S.; Souillac, Pierre O.; Uversky, Vladimir N.

Journal of Biological Chemistry, VOL. 277, NO. 15, April 12, 2002, PP. 112657-12665

Light chain amyloidosis involves the systemic deposition of fibrils in patients overproducing monoclonal immunoglobulin light chains. The kinetics of fibril formation of LEN, a benign light chain variable domain, were investigated at physiological pH in the presence of urea. Despite the lack of in vivo fibril formation, LEN readily forms fibrils in vitro under mildly destabilizing conditions. The effect of low to moderate concentrations of urea on the conformation, association state, stability, and kinetics of fibrillation of LEN were investigated. The conformation of LEN was only slightly affected by the addition of up to 4 M urea. The fibrillation kinetics were highly dependent on protein and urea concentrations, becoming faster with decreasing protein concentration and increasing urea concentration. Changes in spectral probes were concomitant to fibril formation throughout the protein and urea concentration ranges, indicating the absence of off-pathway oligomeric species or amorphous aggregates prior to fibril formation. Reducing the amount of dimers initially present in solution by either decreasing the protein concentration or adding urea resulted in faster fibril formation. Thus, increasing concentrations of urea, by triggering dissociation of dimeric LEN, lead to increased rates of fibrillation.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Chemistry; *Mathematical Biology (Computational Biology); *Methods and Techniques; *attenuated total reflectance-Fourier transform IR spectroscopy ATR-FTIR spectroscopy --Spectrum Analysis Techniques; *small-angle X-ray scattering --X-ray analysis; * FluoroMax-2 fluorescence spectrophotometer --Jobin Yvon-Spex; * FluoroMax-2 fluorescence spectrophotometer --Spectrum Analysis Techniques; *Nicolet 800 spectrophotometer --Nicolet; *Nicolet 800 spectrophotometer --Spectrum Analysis Techniques; *fibril formation dynamics; *immunoglobulin light chain amyloid fibril formation --association state effect; *immunoglobulin light chain amyloid fibril formation --conformational stability effect; *in vitro fibril formation procedure; *light chain amyloidosis; *physiological pH

2002

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli.

Stewart, Richard C.; Boukhvalova, Marina S.; Dahlquist, Frederick W.

Journal of Biological Chemistry, VOL. 277, NO. 25, June 21, 2002, PP. 22251-22259

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant Chew proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *CheA --a protein kinase; *CheW --analysis; *CheW --characterization; *Tar --aspartate receptor; *fluorescence anisotropy --analytical method; *fluorescence anisotropy --fluorometry; *Jobin Yvon-Spex Fluoromax-2 Spectrofluorometer --laboratory equipment; *Jobin Yvon-Spex Fluoromax-2 Spectrofluorometer --Jobin Yvon

2002

Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

Yan, Honggao; Blaszczyk, Jaroslaw; Gong, Yunchen; Ji, Xinhua; Li, Yue; Shi, Genbin

Biochemistry, VOL. 41, NO. 27, July 9, 2002, PP. 8777-8783

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP). Because HPPK is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent. Thermodynamic analysis shows that Mg2+ is important not only for the binding of nucleotides but also for the binding of HMDP. Transient kinetic analysis shows that a step or steps after the chemical transformation are rate-limiting in the reaction catalyzed by HPPK. The pre-steady-state kinetics is composed of a burst phase and a steady-state phase. The rate constant for the burst phase is apprx50 times larger than that for the steady-state phase. The latter is very similar to the kcat value measured by steady-state kinetics. A set of rate constants for the individual steps of the HPPK-catalyzed reaction has been determined by a combination of stopped-flow and quench-flow analyses. These results form a thermodynamic and kinetic framework for dissecting the roles of active site residues in the substrate binding and catalysis by HPPK.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *magnesium(II) ion; *ATP; *6-hydroxymethyl-7,8-dihydropterin; *6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; *fluorometry --analytical method; *fluorometry --photometry; *fluorometry --thermodynamic analysis; *Spex FluoroMax-2 fluorometer --laboratory equipment

2002

Biochemical characterization of two analogues of the apoptosis-linked gene 2 protein in Dictyostelium discoideum and interaction with a physiological partner in mammals, murine Alix.

Klein, Gerard; Aubry, Laurence; Blot, Beatrice; Mattei, Sara; Sadoul, Remy; Satre, Michel

Journal of Biological Chemistry, VOL. 277, NO. 24, June 14, 2002, PP. 21947-21954

Two homologues, Dd-ALG-2a and Dd-ALG-2b, of the mammalian calcium-binding protein ALG-2 (apoptosis-linked gene 2) have been characterized in the cellular slime mold Dictyostelium discoideum. Fluorescence titrations showed that both proteins bind calcium ions with affinities (Ca2+)0.5 of 30 and 450 muM, respectively, at sites specific to calcium. Calcium ion binding resulted in changes of conformation associated with the unmasking of hydrophobic regions of the proteins. Surface plasmon resonance analysis showed that Dd-ALG-2a homodimers formed (KD of 1 muM) at calcium ion concentrations similar to those necessary for Ca2+-induced conformational changes. Deletion of the hydrophobic N-terminal sequence or EF-hand 5 of Dd-ALG-2a prevented dimerization. The Dd-ALG-2b homodimer was not detected, and the Dd-ALG-2a/2b heterodimer formed only when Dd-ALG-2b was the immobilized partner. Murine Alix formed a heterodimer (KD = 0.6 muM) with Dd-ALG-2a but not with Dd-ALG-2b, and the interaction strictly depended upon calcium ions. The DELTANter construct of Dd-ALG-2a lost its interaction capacity with mouse Alix. The genes encoding both proteins, Dd-alg-2a and -2b, were expressed in growing cells. The levels of mRNA were at a maximum during aggregation (4-8 h) and decreased rapidly thereafter. In contrast, the levels of proteins remained fairly stable. Dd-ALG-2a and Dd-ALG-2b were found to be dispensable for growth and development, based on the finding that single Dd-alg2a- or Dd-alg-2b- and double Dd-alg2a-/Dd-alg-2b- mutant cell lines showed normal growth in axenic medium or on bacterial lawns and exhibited unaltered development.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *mammal (Mammalia); *murine (Muridae); *Dictyostelium discoideum (Myxophyta); *Animals; *Chordates; *Fungi; *Mammals; *Microorganisms; *Nonhuman Mammals; *Nonhuman Vertebrates; *Nonvascular Plants; *Plants; *Rodents; *Vertebrates; *apoptosis-linked gene 2 protein --analogue biochemical characterization; *mammalian ALG-2; *Dd-ALG-2a --analogue biochemical characterization; *Dd-ALG-2a --apoptosis-linked gene 2 protein analogue; *Dd-ALG-2b --analogue biochemical characterization; *Dd-ALG-2b --apoptosis-linked gene 2 protein analogue; *AF358911 --amino acid sequence; *AF358911 --nucleotide sequence; *AF358911 --DDBJ; *AF358911 --EBI; *AF358911 --EMBL; *AF358911 --GenBank; *AF358912 --amino acid sequence; *AF358912 --nucleotide sequence; *AF358912 --DDBJ; *AF358912 --EBI; *AF358912 --EMBL; *AF358912 --GenBank; *AF358913 --amino acid sequence; *AF358913 --nucleotide sequence; *AF358913 --DDBJ; *AF358913 --EBI; *AF358913 --EMBL; *AF358913 --GenBank; *AF358914 --amino acid sequence; *AF358914 --nucleotide sequence; *AF358914 --DDBJ; *AF358914 --EBI; *AF358914 --EMBL; *AF358914 --GenBank; *immunofluorescence --Spectrum Analysis Techniques; *surface plasmon resonance analysis --analytical method; *Northern blot --blotting/hybridization/molecular probe techniques; *Northern blot --gene mapping; *Northern blot --labeling; *Northern blot --recombinant DNA technology; *Spex Fluoromax spectrofluorometer --Spectrum Analysis Techniques; *Spex Fluoromax spectrofluorometer --Spex Fluoromax ; *SDS-polyacrylamide gel electrophoresis --polyacrylamide gel electrophoresis; *Western blot --gene mapping; *Western blot --labeling; *apoptosis-linked gene 2 protein-murine Alix interaction; *developmental cycle; *vegetative cycle

2002

An efficient system for the evolution of aminoacyl-tRNA synthetase specificity.

Schultz, Peter G.; Herberich, Brad; King, David S.; Santoro, Stephen W.; Wang, Lei

Nature Biotechnology, VOL. 20, NO. 10, October, 2002, PP. 1044-1048

A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cmr) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E. coli.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae) --expression system; *Bacteria; *Eubacteria; *Microorganisms; *aminoacyl-tRNA synthetase aminoacyl-transfer RNA synthetase --analysis; *aminoacyl-tRNA synthetase aminoacyl-transfer RNA synthetase --substrate specificity evolution; *chloramphenicol resistance reporter --fluorescent marker; *fluorescence-activated cell sorting --analytical method; *fluorescence-activated cell sorting --flow cytometry; *BDIS FACScan instrument --laboratory equipment; *BDIS FACScan instrument --BD Biosciences; *BDIS FACVantage TSO cell sorter --laboratory equipment; *BDIS FACVantage TSO cell sorter --BD Biosciences; * FluoroMax-2 fluorimeter --laboratory equipment