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

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2001

The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif.

Aceto, Antonio; Cocco, Roberta; Dragani, Beatrice; Mannervik, Bengt; Paludi, Domenico; Principe, Daniela Rossi; Stenberg, Gun

Journal of Biological Chemistry, VOL. 276, NO. 34, August 24, 2001, PP. 32177-32183

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this sub-domain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *human glutathione S-transferase A1-1 hGSTA1-1 --alpha class; *human glutathione S-transferase A1-1 hGSTA1-1 --amino-capping box; *human glutathione S-transferase A1-1 hGSTA1-1 --analysis; *human glutathione S-transferase A1-1 hGSTA1-1 --folding; *human glutathione S-transferase A1-1 hGSTA1-1 --mutants; *human glutathione S-transferase A1-1 hGSTA1-1 --stability; *human glutathione S-transferase A1-1 hGSTA1-1 --staple motif; *kinetic analysis --activity assays; *kinetic analysis --analytical method; *site-directed mutagenesis --genetic method; *site-directed mutagenesis --mutagenesis; *site-directed mutagenesis --protein engineering; * Fluoromax spectrofluorometer --laboratory equipment; * Fluoromax spectrofluorometer --Spex; *GeneClean kit --laboratory equipment; *GeneClean kit --BIO 101 Inc.; *Jasco-600 spectropolarimeter --laboratory equipment; *Jasco-600 spectropolarimeter --Jasco

2001

The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase.

Scapozza, Leonardo; Folkers, Gerd; Kessler, Ulrich; Schulz, George E.; Vogt, Joachim; Wurth, Christine

Protein Science, VOL. 10, NO. 1, January, 2001, PP. 63-73

The structure of Herpes simplex virus type 1 thymidine kinase (TKHSV1) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TKHSV1, measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand-free TKHSV1 is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of, TKHSV1 in complex with two new ligands, HPT and HMTT, refined to 2.2 ANG are presented. Although TKHSV1:HPT does not exhibit any significant deviations from the model of TKHSV1:dT, the TKHSV1:HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TKHSV1 substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Herpes simplex virus type 1 (Herpesviridae); *Animal Viruses; *Microorganisms; *Viruses; *nucleoside monophosphate kinase; *thymidine kinase EC 2.7.1.21 --conformational stability; *thymidine kinase EC 2.7.1.21 --crystal structure; *thymidine kinase EC 2.7.1.21 --structural stability; *far-UV circular dichroism --analytical method; *far-UV circular dichroism --Spectrum Analysis Techniques; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *program Origin 5.0 --computer software; *program Origin 5.0 --Inc.; *program Origin 5.0 --MicroCal Software; *protein crystallization --chemical modification; *protein crystallization --crystallization method; *thermal denaturation experiments --analytical method; *thermal denaturation experiments --Molecular Biology Techniques and Chemical Characterization; *CD spectroscopy circular dichroism spectroscopy --analytical method; *CD spectroscopy circular dichroism spectroscopy --spectroscopic techniques: CB; *JASCO J720 spectropolarimeter --laboratory equipment; *JASCO J720 spectropolarimeter --JASCO; *Spex FluoroMax spectrofluorimeter --laboratory equipment; *Spex FluoroMax spectrofluorimeter --Spex; *substrate binding

2001

Supersensitive immunoassay for free PSA using nanoparticle label technology.

Soukka, T.; Paukkunen, J.; Lovgren, T.; Lonnberg, S.; Harma, H.

Clinical Chemistry and Laboratory Medicine, VOL. 39, NO. Special Supplement, 2001, PP. S148

DESCRIPTOR(S)- *Clinical Chemistry (Allied Medical Sciences); *Methods and Techniques; *Oncology (Human Medicine, Medical Sciences); *Urology (Human Medicine, Medical Sciences); *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *prostate specific antigen --free; * Fluoro-Max nanoparticles --delivery system; * Fluoro-Max nanoparticles --particulate labe; *supersensitive immunoassay --immunologic method; *nano-particle label technology; *Meeting Abstract

2001

Structures of apomyoglobin's various acid-destabilized forms.

Callender, Robert H.; Dyer, R. Brian; Gilmanshin, Rudolf; Gulotta, Miriam

Biochemistry, VOL. 40, NO. 17, May 1, 2001, PP. 5127-5136

The structures and the cold and hot melting thermodynamics of the acid- and salt-destabilized states of horse heart apomyoglobin (apoMb), including the E (extended) and various I forms, are studied using probes of tertiary structure (tryptophan fluorescence and FTIR spectroscopy) and secondary structure (far-UV CD and FTIR spectroscopy). These forms likely resemble early structures in the folding of the largely helical protein. Both the I and E forms retain the AGH core whereby the two ends of the protein are tied together with sufficient numbers of tertiary contacts, involving a number of hydrophobic residues, to show cooperative melting. The melting thermodynamics of E and I are distinctly different. E contains no other tertiary structure and probably little other secondary structure apart from the core. The more destabilized E form appears to contain "random" buried runs of polypeptide backbone which convert to alpha-helix in the I form(s). Most interestingly, E consists not of a single structure but is composed of a heterogeneous mixture of conformations, all showing corelike cooperative melting characteristics, and consisting presumably of varying contacts between the A portion of apomyoglobin and the G-H hairpin. These results bear on the energy landscape and structural features of the early part of apomyoglobin's folding pathway.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *apomyoglobin --acid-destabilized forms; *apomyoglobin --analysis; *apomyoglobin --folding pathway; *apomyoglobin --horse heart; *apomyoglobin --secondary structure; *apomyoglobin --tertiary structure; *apomyoglobin --thermodynamics; *far-UV CD spectroscopy far-UV circular dichroism spectroscopy --analytical method; *far-UV CD spectroscopy far-UV circular dichroism spectroscopy --Spectrum Analysis Techniques; *tryptophan fluorescence --analytical method; *tryptophan fluorescence --Spectrum Analysis Techniques; *Bruker model IFS 66 FTIR spectrometer --equipment; *Bruker model IFS 66 FTIR spectrometer --Bruker; * FluoroMax-2 spectrofluorimeter --equipment; * FluoroMax-2 spectrofluorimeter --Instruments S.A. Inc.; *FTIR spectroscopy Fourier transform IR spectroscopy --analytical method; *FTIR spectroscopy Fourier transform IR spectroscopy --IR spectrophotometry: CB; *Jasco J-720 spectrometer --equipment; *Jasco J-720 spectrometer --Jasco

2001

Saposin D solubilizes anionic phospholipid-containing membranes.

Vaccaro, Anna Maria; Arancia, Giuseppe; Ciaffoni, Fiorella; Crateri, Pasqualina; Salvioli, Rosa; Tatti, Massimo

Journal of Biological Chemistry, VOL. 276, NO. 34, August 24, 2001, PP. 31583-31589

Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles. We have presently found that, upon lipid binding, Sap D solubilizes the membranes, as shown by the clearance of the vesicles turbidity. The results of gel filtration, density gradient centrifugation, and negative staining electron microscopy demonstrate that this effect is due to the transformation of large vesicles to smaller particles. The solubilizing effect of Sap D is highly dependent on pH, the lipid/saposin ratio, and the presence of anionic phospholipids; small variations in each of these conditions markedly influences the activity of Sap D. The present study documents the interaction of Sap D with membranes as a complex process. Anionic phospholipids attract Sap D from the medium; when the concentration of the saposin on the lipid surface reaches a critical value, the membrane breaks down into recombinant small particles enriched in anionic phospholipids. Our results suggest that the role played by Sap D is more general than promoting sphingolipid degradation, e.g. the saposin might also be a key mediator of the solubilization of intralysosomal/late endosomal anionic phospholipid-containing membranes.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *vesicle --anionic phospholipid-containing; *anionic phospholipids --analysis; *anionic phospholipids --membrane solubilization; *saposin A Sap A --analysis; *saposin A Sap A --isolation; *saposin A Sap A --lipid binding affinity; *saposin A Sap A --purification; *saposin B Sap B --analysis; *saposin B Sap B --isolation; *saposin B Sap B --lipid binding affinity; *saposin B Sap B --purification; *saposin C Sap C --analysis; *saposin C Sap C --isolation; *saposin C Sap C --lipid binding affinity; *saposin C Sap C --purification; *saposin D Sap D --analysis; *saposin D Sap D --isolation; *saposin D Sap D --lipid binding affinity; *saposin D Sap D --purification; *density gradient centrifugation --centrifugation techniques: CT; *density gradient centrifugation --isolation method; *electrospray mass spectrometry --analytical method; *electrospray mass spectrometry --Spectrum Analysis Techniques; *gel filtration chromatography --purification method; *gel filtration chromatography --Chromatographic Techniques; *negative staining electron microscopy --microscopy method; *negative staining electron microscopy --Imaging Techniques; * FluoroMax-2 spectrofluorimeter --equipment; *Philips 208S electron microscope --equipment; *Philips 208S electron microscope --Philips; *SDS-polyacrylamide gel electrophoresis --separation method; *SDS-polyacrylamide gel electrophoresis --Electrophoretic Techniques

2001

Reversibly bound and covalently attached ligands induce conformational changes in the omega loop, Cys69-Cys96, of mouse acetylcholinesterase.

Taylor, Palmer; Boyd, Aileen E.; Radic, Zoran; Shi, Jianxin

Journal of Biological Chemistry, VOL. 276, NO. 45, November 9, 2001, PP. 42196-42204

We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu76, Glu81, Glu84, Tyr124, Ala262, and His287) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (OMEGA loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the OMEGA loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the OMEGA loop accompany ligand binding.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *mouse (Muridae); *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates; *acetylcholinesterase --analysis; *acetylcholinesterase --characterization; *acrylodan --label; *cysteine-69; *cysteine-96; *kinetic analysis --analytical method; *kinetic analysis --Bioassays/Physiological Analysis; *spectrofluorometry --analytical method; *spectrofluorometry --Spectrum Analysis Techniques; *Jobin Yvon/Spex FluoroMax II spectrofluorometer --laboratory equipment; *Jobin Yvon/Spex FluoroMax II spectrofluorometer --Instrument S.A.

2001

Probing the mechanism of insulin fibril formation with insulin mutants.

Fink, Anthony L.; Brange, Jens; Frokjaer, Sven; Nielsen, Liza; Uversky, Vladimir N.

Biochemistry, VOL. 40, NO. 28, July 17, 2001, PP. 8397-8409

The molecular basis of insulin fibril formation was investigated by studying the structural properties and kinetics of fibril formation of 20 different human insulin mutants at both low pH (conditions favoring monomer/dimer) and at pH 7.4 (conditions favoring tetramer/hexamer). Small-angle X-ray scattering showed insulin to be monomeric in 20% acetic acid, 0.1 M NaCl, pH 2. The secondary structure of the mutants was assessed using far-UV circular dichroism, and the tertiary structure was determined using near-UV circular dichroism, quenching of intrinsic fluorescence by acrylamide and interactions with the hydrophobic probe 1-anilino-8-naphthalene-sulfonic acid (ANS). The kinetics of fibril formation were monitored with the fluorescent dye, Thioflavin T. The results indicate that the monomer is the state from which fibrils arise, thus under some conditions dissociation of hexamers may be rate limiting or partially rate limiting. The insulin mutants were found to retain substantial nativelike secondary and tertiary structure under all conditions studied. The results suggest that fibril formation of the insulin mutants is controlled by specific molecular interactions that are sensitive to variations in the primary structure. The observed effects of several mutations on the rate of fibril formation are inconsistent with a previously suggested model for fibrillation (Brange, J., Whittingham, J., Edwards, D., Youshang, Z., Wollmer, A., Brandenburg, D., Dodson, G., and Finch, J. (1997) Curr. Sci. 72, 470-476). Two surfaces on the insulin monomer are identified as potential interacting sites in insulin fibrils, one consisting of the residues B10, B16, and B17 and the other consisting of at least the residues A8 and B25. The marked increase in the lag time for fibril formation with mutations to more polar residues, as well as mutations to charged residues, demonstrates the importance of both hydrophobic and electrostatic interactions in the initial stages of fibrillation. A model for insulin fibril formation is proposed in which the formation of a partially folded intermediate is the precursor for associated species on the pathway to fibril formation.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *acrylamide; *insulin mutants --Novo Nordisk A/S; *Thioflavin T --fluorescent dye; *Thioflavin T --Sigma; *1-anilino-8-naphthalene-sulfonic acid ANS --hydrophobic probe; *1-anilino-8-naphthalene-sulfonic acid ANS --Sigma; *far-UV circular dichroism --analytical method; *far-UV circular dichroism --Spectrum Analysis Techniques; *near-UV circular dichroism --analytical method; *near-UV circular dichroism --Spectrum Analysis Techniques; *small-angle X-ray scattering --analytical method; *small-angle X-ray scattering --Spectrum Analysis Techniques; *small-angle X-ray scattering instrument SAXS instrument --laboratory equipment; *AVIV 60DS circular dichroism spectrophotometer --laboratory equipment; *AVIV 60DS circular dichroism spectrophotometer --Aviv; * FluoroMax-2 spectrofluorometer --laboratory equipment; * FluoroMax-2 spectrofluorometer --Inc.; * FluoroMax-2 spectrofluorometer --Instruments S. A.; *electrostatic interactions; *hydrophobic interactions; *insulin fibril formation --mechanism; *insulin fibril formation --molecular basis; *intrinsic fluorescence

2001

Physical properties of erythrocyte ghosts that determine susceptibility to secretory phospholipase A2.

Bell, John D.; Harris, Faith M.; Smith, Samantha K.

Journal of Biological Chemistry, VOL. 276, NO. 25, June 22, 2001, PP. 22722-22731

Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A2 (sPLA2) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA2 activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA2 was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *cell membrane; *erythrocyte --blood and lymphatics; *secretory phospholipase A2; *ATP; *electron microscopy --microscopy method; *electron microscopy --microscopy: CB; *electron microscopy --microscopy: CT; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopic techniques: CT; * Fluoromax --equipment; * Fluoromax --Spex Industries; *HPLC --chromatographic techniques; *HPLC --purification method; *HPLC --Isolation/Purification Techniques: CB; *PCR --amplification method; *PCR --DNA amplification; *SDS-PAGE --analytical method; *SDS-PAGE --electrophoretic techniques

2001

Oxygen quenching measurements for the S1 and S2 fluorescence of gas-phase fluoranthene.

Wabuyele, Busolo Wa; Barnes, Russell H.; Ivancic, William A.; Perezzani, Laura; Sassu, Lorenzo

Applied Spectroscopy, VOL. 55, NO. 3, March, 2001, PP. 307-310

The fluorescence emission of the fluoranthene molecule has been investigated at excitation wavelengths varying from 240 to 360 nm, and conducted from near collision-free conditions to atmospheric-pressure conditions. The oxygen quenching rate for the fluorescence of the F1 band (S1fwdarwS0 transition) of fluoranthene at 340 nm excitation wavelength was found to be 1X109 L mol-1 s-1. The quenching rate for the F2 band (S2fwdarwS0 transition) was found to be 100-fold greater for the wavelength region investigated. The wavelength dependency of the oxygen-quenching constants of the two transitions was also established in the excitation range from 240 to 332 nm. The influence of oxygen and nitrogen pressure on the spectral fluorescence bandwidth of fluoranthene and the oxygen-quenching constants for the S1 and S2 bands are reported. The results shed some light on fluoranthene's unusual inertness to oxygen quenching.

DESCRIPTOR(S)- *Methods and Techniques; *Pollution Assessment Control and Management; *fluoranthene molecule --fluorescence emission; *gas-phae fluoranthene --S-1 fluorescence; *gas-phae fluoranthene --S-2 fluorescence; *oxygen quenching measurement --measurement method; *ISA-Spex Fluoromax spectrofluorometer --laboratory equipment; *Stern-Volmer equation --mathematical method; *oxygen-quenching constants --wavelength dependency

2001

Negative cooperativity of substrate binding but not enzyme activity in wild-type and mutant forms of CTP:glycerol-3-phosphate cytidylyltransferase.

Kent, Claudia; Campbell, Heidi A.; Sanker, Subramaniam

Journal of Biological Chemistry, VOL. 276, NO. 41, October 12, 2001, PP. 37922-37928

CTP:glycerol-3-phosphate cytidylyltransferase (GCT) catalyzes the synthesis of CDP-glycerol for teichoic acid biosynthesis in certain Gram-positive bacteria. This enzyme is a model for a cytidylyltransferase family that includes the enzymes that synthesize CDP-choline and CDP-ethanolamine for phosphatidylcholine and phosphatidylethanolamine biosynthesis. We have used quenching of intrinsic tryptophan fluorescence to measure binding affinities of substrates to the GCT from Bacillus subtilis. Binding of either CTP or glycerol-3-phosphate to GCT was biphasic, with two binding constants of about 0.1-0.3 and 20-40 muM for each substrate. The stoichiometry of binding was 2 molecules of substrate/enzyme dimer, so the two binding constants represented distinctly different affinities of the enzyme for the first and second molecule of each substrate. The biphasic nature of binding was observed with the wild-type GCT as well as with several mutants with altered Km or kcat values. This negative cooperativity of binding was also seen when a catalytically defective mutant was saturated with two molecules of CTP and then titrated with glycerol-3-phosphate. Despite the pronounced negative cooperativity of substrate binding, negative cooperativity of enzyme activity was not observed. These data support a mechanism in which catalysis occurs only when the enzyme is fully loaded with 2 molecules of each substrate/enzyme dimer.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Bacillus subtilis (Endospore-forming Gram-Positives); *Bacteria; *Eubacteria; *Microorganisms; *enzyme --activity; *glycerol-3-phosphate --Sigma; *glycerol-3-phosphate cytidylyltransferase GCT ; *substrates --binding affinities; *tryptophan --intrinsic fluorescence; *CTP --lycerol-3-phosphate cytidylyltransferase; *CTP --Sigma; *fluorescence --analytical method; *fluorescence --Spectrum Analysis Techniques; *quenching --analytical method; *quenching --Molecular Biology Techniques and Chemical Characterization; * Fluoromax II spectrofluorimeter --laboratory equipment; * Fluoromax II spectrofluorimeter --Photon Technology Inc.; *Photon Technology Inc. spectrofluorimeter --laboratory equipment; *Photon Technology Inc. spectrofluorimeter --Photon Technology Inc.; *binding stoichiometry; *substrate binding --negative cooperativity

2001

Molecular characterization tissue distribution of a novel member of the S100 family of EF-hand proteins.

Makhatadze, George I.; Gribenko, Alexey V.; Hopper, James E.

Biochemistry, VOL. 40, NO. 51, December 25, 2001, PP. 15538-15548

We have isolated from a human prostate cDNA library a cDNA encoding a novel member of the S100 family of EF-hand proteins. The encoded 99-amino acid protein, designated S100Z, is capable of interacting with another member of the family, S100P. S100Z cDNA was cloned into a bacterial expression system, and the S100Z protein was purified to homogeneity from bacterial lysates by a combination of hydrophobic column and gel-filtration chromatography. Direct amino acid sequencing of the 20 N-terminal amino acids confirmed that the sequence of the recombinant protein is identical to the sequence deduced from the cDNA. Low-resolution structural data have been obtained using circular dichroism and fluorescence spectroscopies, and equilibrium analytical centrifugation. These results show that S100Z is a dimeric, predominantly alpha-helical protein. Addition of calcium to a solution of S100Z changes the fluorescence intensity of the protein, indicating that S100Z is capable of binding calcium ions. Analysis of the calcium-binding isotherm indicates the existence of two calcium-binding sites with apparent affinities on the order of 5X106 and 102 M-1. Binding of calcium results in conformational changes and exposure of hydrophobic surfaces on the protein. Using a PCR-based assay, we have detected differences in the expression level of S100Z mRNA in various tissues. The highest levels were found in spleen and leukocytes. S100Z gene expression appears to be deregulated in some tumor tissues, compared to expression in their normal counterparts.

DESCRIPTOR(S)- *Cell Biology; *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae) --expression system; *Bacteria; *Eubacteria; *Microorganisms; *leukocyte --blood and lymphatics; *leukocyte --immune system; *prostate --reproductive system; *spleen --blood and lymphatics; *spleen --immune system; *calcium --binding sites; *cDNA library; *EF-hand proteins --S100 family; *S100P; *S100Z --tissue distribution; *circular dichroism --imaging method; *circular dichroism --Spectrum Analysis Techniques; *direct amino acid sequencing --sequencing method; *direct amino acid sequencing --Molecular Biology Techniques and Chemical Characterization; *equilibrium analytical centrifugation --separation method; *equilibrium analytical centrifugation --Extraction; *equilibrium analytical centrifugation --Isolation; *equilibrium analytical centrifugation --Purification and Separation Techniques; *fluorescent spectroscopy --imaging method; *fluorescent spectroscopy --Spectrum Analysis Techniques; *gel-filtration chromatography --purification method; *gel-filtration chromatography --Chromatographic Techniques; *hydrophobic column --laboratory equipment; *isoelectric focusing calibration kit --laboratory equipment; *isoelectric focusing calibration kit --Pharmacia; *molecular characterization --characterization method; *molecular characterization --Molecular Biology Techniques and Chemical Characterization; *protein isolation --isolation method; *protein isolation --Extraction; *protein isolation --Isolation; *protein isolation --Purification and Separation Techniques; *protein purification --purification method; *protein purification --Extraction; *protein purification --Isolation; *protein purification --Purification and Separation Techniques; *Beckman XLA ultracentrifuge --laboratory equipment; *Beckman XLA ultracentrifuge --Beckman; * Fluoro-Max spectrofluorimeter --laboratory equipment; *Jasco J-715 automatic recording spectropolarimeter --laboratory equipment; *Jasco J-715 automatic recording spectropolarimeter --Jasco; *PhastGel IEF 3-9 gels --laboratory equipment; *PhastGel IEF 3-9 gels --Pharmacia; *PhastSystem gel electrophoresis system --laboratory equipment; *PhastSystem gel electrophoresis system --Pharmacia; *PCR polymerase chain reaction --detection method; *PCR polymerase chain reaction --in situ recombinant gene expression detection; *PCR polymerase chain reaction --sequencing techniques; *PCR polymerase chain reaction --DNA amplification; *PCR polymerase chain reaction --Molecular Biology Techniques and Chemical Characterization; *PCR-based assay polymerase chain reaction-based assay --laboratory equipment; *QIAquick PCR purification kit --laboratory equipment; *QIAquick PCR purification kit --Qiagen

2001

Localization of the chaperone domain of FKBP52.

Buchner, Johannes; Fischer, Elke; Modrow, Susanne; Pirkl, Franziska

Journal of Biological Chemistry, VOL. 276, NO. 40, October 5, 2001, PP. 37034-37041

FKBP52, a multidomain peptidyl prolyl cis/trans-isomerase (PPIase), is found in complex with the chaperone Hsp90 and the co-chaperone p23. It displays both PPIase and chaperone activity in vitro. To localize these two activities to specific regions of the protein, we created and analyzed a set of fragments of FKBP52. The PPIase activity toward both peptides and proteins is confined entirely to domain 1 (amino acids 1-148). The chaperone activity, however, resides in the C-terminal part of FKBP52, mainly in the region between amino acids 264 and 400 (domain 3). Interestingly, this domain also contains the tetratricopeptide repeats, which are responsible for the binding to C-terminal amino acids of Hsp90. Competition assays with a C-terminal Hsp90 peptide suggest that the non-native protein and Hsp90 are bound by different regions within this domain.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Equipment, Apparatus, Devices and Instrumentation; *Methods and Techniques; *chaperone Hsp90; *co-chaperone p23; *tetratricopeptide repeats; *FKBP52 --chaperone activity; *FKBP52 --multidomain peptidyl prolyl cis/trans-isomerase; *FKBP52 chaperone domain --localization; *competition assay --analytical method; *competition assay --Bioassays/Physiological Analysis; *JascoV-550 UV-visable spectrophotometer --laboratory equipment; *Spex Fluoromax-1 fluorescence spectrometer --laboratory equipment; *Spex Fluoromax-1 fluorescence spectrometer --Spex Industries; *Spex Fluoromax-2 fluorescence spectrometer --laboratory equipment; *Spex Fluoromax-2 fluorescence spectrometer --Spex Industries

2001

Interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with the cationic and zwitterionic forms of the fluorescent substrate N(7)-methylguanosine.

Kierdaszuk, Borys; Shugar, David; Stoychev, Gerasim

Biochimica et Biophysica Acta, VOL. 1544, NO. 1-2, 12 January, 2001, PP. 74-88

Steady-state and time-resolved fluorescence spectroscopy, and enzyme kinetics, were applied to study the reaction of purine nucleoside phosphorylase (PNP) from Escherichia coli with its substrate N(7)-methylguanosine (m7Guo), which consists of an equilibrium mixture of cationic and zwitterionic forms (pKa = 7.0), each with characteristic absorption and fluorescence spectra, over the pH range 6-9, where absorption and intrinsic fluorescence of the enzyme are virtually unchanged. The pH-dependence of kinetic constants for phosphorolysis of m7Guo were studied under condition where the population of the zwitterion varied from 10% to 100%. This demonstrated that, whereas the zwitterion is a 3- to 6-fold poorer substrate, if at all, than the cation for the mammalian enzymes, both ionic species are almost equally good substrates for E. coli PNP. The imidazole-ring-opened form of m7Guo is neither a substrate nor an inhibitor of phosphorolysis. Enzyme fluorescence quenching, and concomitant changes in absorption and fluorescence spectra of the two ionic species of m7Guo on binding, showed that both forms are bound by the enzyme, the affinity of the zwitterion being 3-fold lower than that of the cation. Binding of m7Guo is bimodal, i.e., an increase in ligand concentration leads to a decrease in the association constant of the enzyme-ligand complex, typical for negative cooperativity of enzyme-ligand binding, with a Hill constant < 1. This is in striking contrast to interaction of the enzyme with the parent Guo, for which the association constant is independent of concentration. The weakly fluorescent N(7)-methylguanine (m7Gua), the product of phosphorolysis of m7Guo, is a competitive non-substrate inhibitor of phosphorolysis (Ki = 8 +- 2 muM) and exhibits negative cooperativity on binding to the enzyme at pH 6.9. Quenching of enzyme emission by the ligand is a static process, inasmuch as the mean excited-state lifetime, ltbbractaurtbbrac = 2.7 ns, is unchanged in the presence of the ligands, and the constants KSV may therefore be considered as the association constants for the enzyme-ligand complexes. In the pH range 9.5-11 there is an instantaneous reversible decrease in PNP emission of apprx 15%, corresponding to one of the six tyrosine residues per subunit readily accessible to solvent, and OH- ions. Relevance of the overall results to the mechanism of phosphorolysis, and binding of substrates/inhibitors is discussed.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *purine nucleoside phosphorylase --interactions; *N(7)-methylguanosine --cationic form; *N(7)-methylguanosine --enzyme substrate; *N(7)-methylguanosine --zwitterionic form; *affinity chromatography --liquid chromatography; *affinity chromatography --purification method; *fluorescence quenching --detection method; *fluorescence quenching --detection/labeling techniques; *protein purification --purification method; *protein purification --Isolation/Purification Techniques: CB; *x-ray crystallography --imaging method; *x-ray crystallography --X-ray analysis; *Spex FluoroMax spectrofluorometer --equipment; *Spex FluoroMax spectrofluorometer --Spex; *protein structure

2001

HU binding to DNA: Evidence for multiple complex formation and DNA bending.

Mukerji, Ishita; Cole, James L.; Hawkins, Mary E.; Wojtuszewski, Kristi

Biochemistry, VOL. 40, NO. 8, February 27, 2001, PP. 2588-2598

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (apprx1 X 106 M-1), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae) --strain-RLM1078; *Bacteria; *Eubacteria; *Microorganisms; *DNA --analysis; *DNA --bending; *DNA --multiple complex formation; *DNA --HU binding; *HU --analysis; *HU --histone-like DNA binding protein; *3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin --fluorescence probe; *analytical ultracentrifugation --analytical method; *analytical ultracentrifugation --Molecular Biology Techniques and Chemical Characterization; *fluorescence anisotropy --analytical method; *fluorescence anisotropy --Molecular Biology Techniques and Chemical Characterization; *gel mobility shift assay --bioassay method; *gel mobility shift assay --Bioassays/Physiological Analysis; * FluoroMax-2 fluorometer --equipment; * FluoroMax-2 fluorometer --Instruments SA

2001

Homogeneous fluoroimmunoassay of a pyrethroid metabolite in urine.

Hammock, Bruce D.; Gee, Shirley J.; Kennedy, Ian M.; Matveeva, Evgenia G.; Shan, Guomin; Stoutamire, Donald W.

Analytica Chimica Acta, VOL. 444, NO. 1, 12 October, 2001, PP. 103-117

Pyrethroids are widely used in agriculture as insecticides. In this study, we describe a simple one-step homogeneous fluoroimmunoassay for the glycine conjugate of phenoxybenzoic acid (PBAG), a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Quenching fluoroimmunoassay (QFIA) is based on the competition of labeled and non-labeled pesticide for binding with antibodies and the resulting calibration curve is based on the relationship between analyte concentration and fluorescence quenching of labeled pesticide by specific antibodies. We developed a QFIA for PBAG in aqueous solution using fluorescein-labeled PBAG and polyclonal antibodies. The estimated IC50 (analyte concentration giving 50% inhibition of quenching) for PBAG was 4.5 nM. The detection limit (DL) was 0.9 nM. The dynamic range of the calibration curve was 2-50 nM. The average analytical recovery obtained by applying the method to urine samples (400- or 1000-fold urine dilution.) was 85-111%. This demonstrates the QFIA to be a very simple and rapid detection method for PBAG; no washing steps and no enzyme conjugates were required.

DESCRIPTOR(S)- *Clinical Chemistry (Allied Medical Sciences); *Methods and Techniques; *Pesticides; *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *urine --excretory system; *phenoxybenzoic acid --pyrethroid exposure biomarker; *phenoxybenzoic acid --pyrethroid metabolite; *phenoxybenzoic acid --urine content; *polyclonal antibodies; *fluorometer --Fluoromax II spectrofluorometer; *fluorometer --Jobin Yvon-Spex; *quenching fluoroimmunoassay --detection method; *quenching fluoroimmunoassay --labeling

2001

Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta2 adrenergic receptor.

Kobilka, Brian K.; Farrens, David L.; Ghanouni, Pejman; Gryczynski, Zygmunt; Lakowicz, Joseph R.; Lee, Tae Weon; Steenhuis, Jacqueline J.

Journal of Biological Chemistry, VOL. 276, NO. 27, July 6, 2001, PP. 24433-24436

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta2 adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta2 adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Pharmacology; *beta-2 adrenergic receptor --analysis; *G protein-coupled receptors; *spectrofluorometry --analytical method; *spectrofluorometry --spectroscopic techniques: CB; *SPEX Fluoromax spectrofluorometer --equipment

2001

Folding and self-assembly of herpes simplex virus type 1 thymidine kinase.

Scapozza, Leonardo; Folkers, Gerd; Thomas, Richard M.; Wurth, Christine

Journal of Molecular Biology, VOL. 313, NO. 3, 26 October, 2001, PP. 657-670

Thymidine kinase from herpes simplex virus type 1 (HSV1 TK) has been postulated to be a homodimer throughout the X-ray crystallography literature. Our study shows that HSV1 TK exists as a monomer-dimer equilibrium mixture in dilute aqueous solutions. In the presence of 150 mM NaCl, the equilibrium is characterized by a dissociation constant of 2.4 muM; this constant was determined by analytical ultracentrifugation and gel filtration experiments. Dimerization seems to be unfavorable for enzymatic activity: dimers show inferior catalytic efficiency compared to the monomers. Moreover, soluble oligomers formed by self-assembly of TK in the absence of physiological salt concentrations are even enzymatically inactive. This study investigates enzymatic and structural relevance of the TK dimer in vitro. Dissociation of the dimers into monomers is not accompanied by large overall changes in secondary or tertiary structure as shown by thermal and urea-induced unfolding studies monitored by circular dichroism and fluorescence spectroscopy. A disulfide-bridge mutant TK (V119C) was designed bearing two cysteine residues at the dimer interface in order to crosslink the two subunits covalently. Under reducing conditions, the properties of V119C and wild-type HSV1 TK (wt HSV1 TK) were identical in terms of expression yield, denaturing SDS PAGE gel electrophoresis, enzyme kinetics, CD spectra and thermal stability. Crosslinked V119C (V119Cox) was found to have an increased thermal stability with a tm value of 59.1(+-0.5)degreeC which is 16 deg. C higher than for the wild type protein. This is thought to be a consequence of the conformational restriction of the dimer interface. Furthermore, enzyme kinetic studies on V119Cox revealed a Km for thymidine of 0.2 muM corresponding to wt HSV1 TK, but a significantly higher Km for ATP. The present findings raise the question whether the monomer, not the dimer, might be the active species in vivo.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *herpes simplex virus type 1 thymidine kinase --folding; *herpes simplex virus type 1 thymidine kinase --self-assembly; *analytical ultracentrifugation --centrifugation; *analytical ultracentrifugation --separation method; *circular dichroism --analytical method; *circular dichroism --Spectrum Analysis Techniques; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --spectroscopy; *protein engineering --genetic engineering; *protein engineering --molecular genetic method; *size-exclusion chromatography --separation method; *size-exclusion chromatography --Chromatographic Techniques; *Jasco J720 spectropolarimeter --equipment; *Jasco J720 spectropolarimeter --Jasco; *Spex FluoroMax spectrofluorimeter --equipment; *Superdex 200 HR 10/30 FPLC column --equipment; *Superdex 200 HR 10/30 FPLC column --Pharmacia; *X-ray crystallography --analytical method; *X-ray crystallography --X-ray analysis; *functional state; *molecular evolution

2001

Factors underlying membrane potential-dependent and -independent fluorescence responses of potentiometric dyes in stressed cells: diS-C3(3) in yeast.

Gaskova, D.; Cadek, R.; Chaloupka, R.; Plasek, J.; Sigler, K.

Biochimica et Biophysica Acta, VOL. 1511, NO. 1, 9 March, 2001, PP. 74-79

The redistribution fluorescent dye diS-C3(3) responds to yeast plasma membrane depolarisation or hyperpolarisation by DELTAPSI-dependent outflow from or uptake into the cells, reflected in changes in the fluorescence maximum lambdamax and fluorescence intensity. Upon membrane permeabilisation the dye redistributes between the cell and the medium in a purely concentration-dependent manner, which gives rise to DELTAPSI-independent fluorescence responses that may mimic DELTAPSI-dependent blue or red shift in lambdamax. These lambdamax shifts after cell permeabilisation depend on probe and ion concentrations inside and outside the cells at the moment of permeabilisation and reflect (a) permeabilisation-induced DELTAPSI collapse, (b) changing probe binding capacity of cell constituents (inverse to the ambient ionic strength) and (c) hampering of probe equilibration by the poorly permeable cell wall. At low external ion concentrations, cell permeabilisation causes ion outflow and probe influx (hyperpolarisation-like red shift in lambdamax) caused by an increase in the probe-binding capacity of the cell interior and, in the case of heat shock, protein denaturation unmasking additional probe-binding sites. At high external ion levels minimising net ion efflux and at high intracellular probe concentrations at the moment of permeabilisation, the DELTAPSI collapse causes a blue lambdamax shift mimicking an apparent depolarisation.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Membranes (Cell Biology); *Methods and Techniques; *Saccharomyces cerevisiae (Ascomycetes); *Fungi; *Microorganisms; *Nonvascular Plants; *Plants; *potentiometric dyes --membrane potential-dependent fluorescence responses; *potentiometric dyes --membrane-independent fluorescence responses; *spectrofluorimetry --FluoroMax 2; *spectrofluorimetry --Spectrum Analysis Techniques; *biomembranes; *cell compartment binding probes; *permeabilization; *stressed cell factors

2001

Effect of familial Parkinson's disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human alpha-synuclein.

Fink, Anthony L.; Li, Jie; Uversky, Vladimir N.

Biochemistry, VOL. 40, NO. 38, September 25, 2001, PP. 11604-11613

Parkinson's disease involves the loss of dopaminergic neurons in the substantia nigra, leading to movement disorders. The pathological hallmark of Parkinson's disease is the presence of Lewy bodies and Lewy neurites, which are intracellular inclusions consisting primarily of alpha-synuclein. Although essentially all cases of sporadic and early-onset Parkinson's disease are of unknown etiology, two point mutations (A53T and A30P) in the alpha-synuclein gene have been identified in familial early-onset Parkinson's disease. Previous reports have shown that mutant alpha-synuclein may form fibrils more rapidly than wild-type protein. To determine the underlying molecular basis for the enhanced fibrillation of the mutants, the structural properties, responses to changes in the environment, and propensity to aggregate of wild-type, A30P, and A53T alpha-synucleins were systematically investigated. A variety of biophysical methods, including far-UV circular dichroism, FTIR, small-angle X-ray scattering, and light scattering, were employed. Neither the natively unfolded nor the partially folded intermediate conformations are affected by the familial Parkinson's disease point mutations. However, both mutants underwent self-association more readily than the wild type (i.e., at much lower protein concentration and more rapidly). We attribute this effect to the increased propensity of their partially folded intermediates to aggregate, rather than to any changes in the monomeric natively unfolded species. This increased propensity of these mutants to aggregate, relative to wild-type alpha-synuclein, would account for the correlation of these mutations with Parkinson's disease.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Nervous System (Neural Coordination); *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *Parkinson's disease --nervous system disease; *alpha-synuclein --analysis; *far-UV circular dichroism --analytical method; *far-UV circular dichroism --spectroscopic techniques: CB; *light scattering --analytical method; *light scattering --Bioassays/Physiological Analysis; *small-angle X-ray scattering --analytical method; *small-angle X-ray scattering --X-ray analysis; *AVIV 60DS spectrophotometer --equipment; *AVIV 60DS spectrophotometer --AVIV; * FluoroMax-2 spectrofluorometer --equipment; * FluoroMax-2 spectrofluorometer --Instruments S.A.; *FTIR spectroscopy Fourier transform IR spectroscopy --analytical method; *FTIR spectroscopy Fourier transform IR spectroscopy --IR spectrophotometry: CB; *Nicolet 800SX Fourier transform IR spectrometer --equipment; *Nicolet 800SX Fourier transform IR spectrometer --Nicolet; *Parkinson Disease (MeSH)

2001

DNA condensation by polyamines: A laser light scattering study of structural effects.

Thomas, T. J.; Shirahata, Akira; Thomas, Thresia; Vijayanathan, Veena

Biochemistry, VOL. 40, NO. 45, November 13, 2001, PP. 13644-13651

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH-(CH2)3NH2 (n=4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4+-1 muM) for spermine homologues with n=4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log (EC50(polyamine4+)) against log (Na+) was apprx1.5 for polyamines with even number values of n, whereas the slope value was apprx1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n=3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh=60-70 nm for n=7-10 and >100 nm for n=11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n=2 and 3 as well as those with n>7 had significantly lower DNA binding affinity compared to spermine and homologues with n=5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *lambda-DNA --analysis; *lambda-DNA --Sigma; *polyamines; *sodium cacodylate --buffer; *spermidine; *spermine hydrochloride --Sigma; *dynamic light scattering measurement --measurement method; *dynamic light scattering measurement --Spectrum Analysis Techniques; * Fluoromax-2 spectrofluorometer --equipment; * Fluoromax-2 spectrofluorometer --Jobin Yvon-Spex Instruments

2001

Development of sensitive esterase assays based on alpha-cyano-containing esters.

Shan, Guomin; Hammock, Bruce D.

Analytical Biochemistry, VOL. 299, NO. 1, December 1, 2001, PP. 54-62

A novel approach is reported for the development of fluorogenic esterase reporters using alpha-cyano-containing esters as substrates. After ester hydrolysis, the released alcohol, a cyanohydrin, rapidly eliminates HCN to yield the corresponding aldehyde resulting in strong fluorescence. The pi conjugation of the resulting aldehyde also greatly enhances UV absorption and red shifts fluorescence emission relative to a corresponding alcohol or phenol. Two substrates, R/S-acetic acid cyano-(6-methoxynaphthalen-2-yl)-methyl ester (compound I) and trans/cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid R/S-cyano-(6-methoxynaphthalen-2-yl)-methyl ester (compound II), were synthesized and evaluated as substrates. Such alpha-cyano substrates possess very low background fluorescence and are more stable under enzyme assay conditions than phenolic substrates due to the aliphatic cyano group. The higher molar absorbtivity and quantum yield of the aldehyde, along with its larger Stokes' shift combined with the increased stability and lower background signal of the cyanohydrin substrate, increases the utility and sensitivity of the resulting assays over current methods. Moreover, compound II showed high selectivity to pyrethroid-cleaving esterases and may provide a direct tool to monitor pyrethroid resistance in insects.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *esterase --assay; *trans/cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid R/S-cyano-(6-methoxynaphthalen-2-yl)methyl ester --substrate; *R/S-acetic acid cyano-(6-methoxynaphthalen-2-yl)-methyl ester --substrate; *esterase assay --analytical method; *esterase assay --Bioassays/Physiological Analysis; *fluorospectrometry --analytical method; *fluorospectrometry --Spectrum Analysis Techniques; * Fluoromax-2 fluorospectometer --laboratory equipment; * Fluoromax-2 fluorospectometer --Instruments S. A.

2001

Characterization of Bis-8-hydroxyquinoline-Armed Diazatrithia-16-crown-5 and Diazadibenzo-18-crown-6 Ligands as Fluorescent Chemosensors for Zinc

Jun Kawakami, R. Todd Bronson, Guoping Xue, Jerald S. Bradshaw,Reed M. Izatt and Paul B. Savage

Journal of Supramolecular Chemistry 1 (2001) 221-227

 

2001

Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin / DREAM / KChIP3.

Ames, James B.; Buxbaum, Joseph D.; Cheng, H.-Y. Mary; Ikura, Mitsuhiko; Lilliehook, Christina; Osawa, Masanori; Penninger, Josef M.; Tong, Kit I.; Wasco, Wilma

Journal of Biological Chemistry, VOL. 276, NO. 44, November 2, 2001, PP. 41005-41013

Calsenilin/DREAM/KChIP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca2+ binding measurements indicate that the protein binds 3 Ca2+ with a dissociation constant of 14 muM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca2+-bound protein exists as a dimer at protein concentrations lower than 150 muM and forms a tetramer at concentrations above 200 muM. The Ca2+-free protein is a tetramer in the concentration range 20-450 muM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca2+-free protein tetramer binds endothermically (DELTAH=+25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (Kd) of 75 nM, and the other three bind more weakly (Kd=640 nM). No significant DNA binding was observed for the Ca2+-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca2+-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (Kd=200 nM) in a Ca2+-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca2+-free state and dissociates into dimers at saturating Ca2+ levels.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Nervous System (Neural Coordination); *human (Hominidae); *Animals; *Chordates; *Humans; *Mammals; *Primates; *Vertebrates; *neuron --nervous system; *calcium; *calsenilin DREAM, KChIP3 --neuronal calcium-sensing protein; *calsenilin DREAM, KChIP3 --oligomerization; *downstream regulatory element; *presenilins; *prodynorphin; *DNA --binding activity; *dynamic light scattering --identification method; *dynamic light scattering --Spectrum Analysis Techniques; *fast protein liquid chromatography --assessment method; *fast protein liquid chromatography --Chromatographic Techniques; *fluorescence spectroscopy --assessment method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *fluorescent EMSA fluorescent electrophoretic mobility shift assay --identification method; *fluorescent EMSA fluorescent electrophoretic mobility shift assay --restriction fragment mapping; *fluorescent EMSA fluorescent electrophoretic mobility shift assay --Molecular Biology Techniques and Chemical Characterization; *physiochemical characterization --characterization method; *physiochemical characterization --Molecular Biology Techniques and Chemical Characterization; *size exclusion chromatography --identification method; *size exclusion chromatography --liquid chromatography; *size exclusion chromatography --Chromatographic Techniques; *ultracentrifugation --centrifugation; *ultracentrifugation --separation method; *ultracentrifugation --Extraction; *ultracentrifugation --Isolation; *ultracentrifugation --Purification and Separation Techniques; *Beckman model TJ-6 tabletop centrifuge --laboratory equipment; *Beckman model TJ-6 tabletop centrifuge --Beckman; *CD spectroscopy --assessment method; *CD spectroscopy --Spectrum Analysis Techniques; *DNA synthesizer --laboratory equipment; * FluoroMax-2 fluorometer --laboratory equipment; *J-270 CD spectropolarimeter --laboratory equipment; *J-270 CD spectropolarimeter --JASCO; *MicroCal VP-TIC MicroCalorimeter --laboratory equipment; *MicroCal VP-TIC MicroCalorimeter --MicroCal Inc.; *PD-10 gel filtration column --laboratory equipment; *PD-10 gel filtration column --Amersham Pharmacia Biotech; *Storm 860 system --laboratory equipment; *Storm 860 system --Molecular Dynamics; *Superdex 200 HR 10/30 column --laboratory equipment; *Superdex 200 HR 10/30 column --Amersham Pharmacia Biotech; *Zorbax oligonucleotide column --laboratory equipment; *Zorbax oligonucleotide column --DuPont; *dissociation; *Hill coefficient

2001

Ca2+-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

Collins, John H.; Kobayashi, Minae; Kobayashi, Tomoyoshi

Biochimica et Biophysica Acta, VOL. 1594, NO. 2, 18 October, 2001, PP.148-154

to measure Ca2+-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca2+, resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 ANG to 44.1 ANG when Ca2+ bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca2+. The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of apprx0.1 in the presence of Ca2+, and apprx0.3 in the absence of Ca2+, were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by apprx7 ANG in the presence of Ca2+ and by apprx2 ANG in the absence of Ca2+ when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.

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; *thin filaments --muscular system; *actin --analysis; *calcium (II) ion --analysis; *cysteines --analysis; *myosin subfragment 1 myosin S1 --analysis; *tropomyosin Tm --analysis; *troponin C TnC --analysis; *troponin C TnC --natural; *troponin C TnC --regulatory sites; *troponin I TnI --analysis; *troponin I TnI --inhibitory region; *troponin I TnI --mutant; *troponin T TnT --analysis; *troponin T TnT --natural; *troponin-tropomyosin complex Tn-Tm complex --analysis; *N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine IAEDANS --fluorescent probe; *4-dimethylaminophenylazophenyl-4'-maleimide DABmal --fluorescent probe; *fluorescence resonance energy transfer FRET --measurement method; *fluorescence resonance energy transfer FRET --Spectrum Analysis Techniques; *S.A. FluoroMax-2 spectrometer --laboratory equipment; *S.A. FluoroMax-2 spectrometer --JOBIN YVON-Spex; *energy transfer efficiency; *proximity changes; *spatial rearrangements

2001

Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme.

Benkovic, Stephen J.; Alley, Stephen C.; Ishmael, Faoud T.; Jones, A. Daniel; Mayer, M. Uljana; Trakselis, Michael A.

Journal of Biological Chemistry, VOL. 276, NO. 42, October 19, 2001, PP. 39340-39349

Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *bacteriophage T4 (Myoviridae); *Bacterial Viruses; *Microorganisms; *Viruses; *bacteriophage T4 DNA polymerase holoenzyme; *DNA; *analytical ultracentrifugation --separation method; *analytical ultracentrifugation --Extraction; *analytical ultracentrifugation --Isolation; *analytical ultracentrifugation --Purification and Separation Techniques; *fluorescence resonance energy transfer analysis --detection method; *fluorescence resonance energy transfer analysis --Spectrum Analysis Techniques; *isothermal titration calorimetry --detection method; *isothermal titration calorimetry --Molecular Biology Techniques and Chemical Characterization; *mass spectrometry --detection method; *mass spectrometry --Spectrum Analysis Techniques; *microcentrifuge tubes --laboratory equipment; *model 1100 HPLC system --laboratory equipment; *model 1100 HPLC system --Hewlett-Packard; *photocross-linking --detection method; *photocross-linking --Spectrum Analysis Techniques; *sliding clamp --laboratory equipment; *Beckman HPLC system --laboratory equipment; *Beckman HPLC system --Beckman; *Beckman Instruments XL-I Analytical Ultracentrifuge --laboratory equipment; *Beckman Instruments XL-I Analytical Ultracentrifuge --Beckman; *BetaBasic C18 column --laboratory equipment; *BetaBasic C18 column --Keystone Scientific; *C18 column --laboratory equipment; * Fluoro-Max-2 spectrofluorimeter --laboratory equipment; *Mariner mass spectrometer --laboratory equipment; *Mariner mass spectrometer --Perspective Biosystems; *Mono-Q anion-exchange column --laboratory equipment; *VP-ITC MicroCalorimeter --laboratory equipment; *VP-ITC MicroCalorimeter --Microcal; *protein-protein interactions

2001

Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter.

Gether, Ulrik; Carroll, F. Ivy; Jensen, Anne Dam; Maresch, Martin J.; Rasmussen, Soren G. F.; Tate, Christopher G.

Journal of Biological Chemistry, VOL. 276, NO. 7, February 16, 2001, PP. 4717-4723

To explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3beta-(4-methylphenyl)tropane-2beta-carboxylic acid N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester hydrochloride (RTI-233); KI = 14 nM) that contained the environmentally sensitive fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized. Specific binding of RTI-233 to the rat serotonin transporter, purified from Sf-9 insect cells, was demonstrated by the competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55 (2beta-carbomethoxy-3beta-(4-iodophenyl)tropane). Moreover, specific binding was evidenced by measurement of steady-state fluorescence anisotropy, showing constrained mobility of bound RTI-233 relative to RTI-233 free in solution. The fluorescence of bound RTI-233 displayed an emission maximum (lambdamax) of 532 nm, corresponding to a 4-nm blue shift as compared with the lambdamax of RTI-233 in aqueous solution and corresponding to the lambdamax of RTI-233 in 80% dioxane. Collisional quenching experiments revealed that the aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (KSV = 1.7 M-1), although not to the same extent as free RTI-233 (KSV = 7.2 M-1). Conversely, the hydrophobic quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous quenching and, thus, partially exposed to solvent.

DESCRIPTOR(S)- *Nervous System (Neural Coordination); *citalopram; *cocaine --binding; *serotonin; *serotonin transporter --biophysical characterization; *serotonin transporter --cocaine binding pocket; *RTI-233 --fluorescent cocaine analog; *RTI-233 --molecular reporter; *RTI-55; *7-nitrobenzo-2-oxa-1,3-diazole NBD --fluorescent moiety; *fluorescence anisotropy --analytical method; *fluorescence anisotropy --Bioassays/Physiological Analysis; *SPEX fluoromax-2 fluorometer --laboratory equipment; *SPEX fluoromax-2 fluorometer --SPEX

2001

A molecular beacon strategy for the thermodynamic characterization of triplex DNA: Triplex formation at the promoter region of cyclin D1.

Thomas, T. J.; Antony, Thomas; Shirahata, Akira; Sigal, Leonard H.; Thomas, Thresia

Biochemistry, VOL. 40, NO. 31, August 7, 2001, PP. 9387-9395

We studied the formation of triplex DNA in the purine-pyrimidine-rich promoter site sequence of cyclin D1, located at -116 to -99 from the transcription initiation site, with a molecular beacon comprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. Formation of triplex DNA was monitored by enhanced fluorescence of the beacon, due to the weakening of fluorescence energy transfer, upon its binding to the target duplex. Electrophoretic mobility shift assay confirmed triplex DNA formation by these oligonucleotides. In low salt buffer (10 mM Na+), triplex DNA formation was not observed in the absence of a ligand such as spermine. At room temperature (22degreeC), the equilibrium association constant (Ka) calculated in the presence of 1 muM spermine and 10 mM Na+ was 3.2X108 M-1. The Ka value was 1.0X109 M-1 in the presence of 150 mM Na+, and it increased by 10-fold by the addition of 1 mM spermine. DELTAH, DELTAS, and DELTAG of triplex DNA formation, calculated from the temperature dependence of Ka in the range of 20-45degreeC, were -35.9 kcal/mol, -77 cal/(molcntdotK), and -13 kcal/mol, respectively, in the presence of 150 mM NaCl. The corresponding values were -52.9 kcal/mol, -132.5 cal/(molcntdotK), and -13.4 kcal/mol in the presence of 150 mM NaCl and 1 mM spermine. Structurally related polyamines exerted different degrees of triplex DNA stabilization, as determined by binding constant measurements. Comparison of spermine versus hexamine showed a 17-fold increase in the equilibrium association constant, whereas bis(ethyl) derivatization lead to a 4-fold decrease of this value. In the absence of added duplex and polyamines, the molecular beacon dissociated with a melting temperature of 67degreeC. Thermodynamic parameters of beacon melting were calculated from the melting curve, and the DELTAH, DELTAS, and DELTAG values were 37.8 kcal/mol, 112 cal/(molcntdotK), and 4.4 kcal/mol, respectively. These results demonstrate that molecular beacons can be used for the direct determination of the equilibrium association constants and thermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *cyclin DNA --promoter region; *oligodeoxyribonucleotide; *triplex DNA --thermodynamic characterization; *electrophoretic mobility shift assay --molecular genetic method; *electrophoretic mobility shift assay --restriction fragment mapping; *fluorescence resonance energy transfer --analytical method; *fluorescence resonance energy transfer --Analysis/Characterization Techniques: CB; * Fluoromax-2 spectrofluorometer --equipment; * Fluoromax-2 spectrofluorometer --Instruments SA; *molecular beacon strategy; *triplex formation