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

タイトル

著者

掲載誌

概要

1998

Validation of a high throughput screen to detect potential modulators of Ca-2+ release-activated Ca-2+ channels in Jurkat T-cells.

Bridgland-Taylor, M. H.; Dale, I. L.; Alexander, P. D.; Tucker, E.; Midha, A.; Randall, V. A.; Sullivan, E.; Pollard, C. E.

British Journal of Pharmacology, VOL. 125, NO. PROC. SUPPL.1998, 82P page(s)

DESCRIPTOR(S)- *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CALCIUM ION; *CALCIUM ION CHANNEL; *CALCIUM RELEASE-ACTIVATED; *CELL BIOLOGY; *DETECTION METHOD; *EXTRACELLULAR CHELATION; *FLIPR ASSAY; *FLUORIMETRY; * FLUOROMAX ASSAY; *HIGH THROUGHPUT; *HUMAN T-CELLS; *INTRACELLULAR; *JURKAT CELL LINE; *LOW THROUGHPUT; *MEETING POSTER; *METHODOLOGY; *MODULATION; *SCREENING METHOD; *THAPSIGARGIN; *VALIDATION

1998

Unfolding of the tetrameric loop deletion mutant of ROP protein is a second-order reaction.

Lassalle, M. W.; Hinz, H-J.

Biochemistry, VOL. 37, NO. 23, 1998, PP. 8465-8472

Comprehensive kinetic studies were carried out on the unfolding properties of RM6 as a function of GdnHCl concentration and temperature. This protein is a mutant resulting from the dimeric wild-type CoLE1-ROP protein by deletion of 5 amino acids (Asp 30, Ala 31, Asp 32, Glu 33, Gln 34) in the loop of each monomer. The deletion has dramatic consequences. The dimeric 4-alpha.-helix structure characteristic of the wild-type protein is completely reorganized and the RM6 structure can be described as a tetrameric alpha helix of extended monomers without loops. These extraordinary structural changes are accompanied by an enormous increase in transition temperature from 71 to 101 degree C. These features have been discussed in a separate publication (1). The remarkable change in thermal stability of RM6 should be reflected in significant changes in the folding rate constants. This was observed in the present unfolding studies. Decay of tetrameric RM6 was monitored by circular dichroism (CD) and fluorescence to probe for changes in both secondary and tertiary structure, respectively. The identity of the kinetic parameters obtained from the two techniques supports the view that secondary and tertiary structure break down simultaneously. However, the most intriguing result is the finding that unfolding of tetrameric RM6 can be described very well by a second-order reaction. The magnitude of the second-order rate constant k-2 varies dramatically with both temperature and denaturant concentration. At 25 degree C and 6.5 M GdnHCl concentration k-2 is 4200 L cntdot (mol of dimer)-1 cntdot s-1, whereas at 4.4 M GdnHCl a value of k-2= 0.9 L cntdot (mol of dimer)-1 cntdot s-1 is observed. Correspondingly, apparent activation enthalpies show a strong increase from DELTA-H = 29.1 kJ cntdot mol-1 at 6.5 M GdnHCl to DELTA-H = 79.7 kJ cntdot mol-1 at 4.4 M GdnHCl. A mechanism involving a dimeric intermediate is suggested which permits a consistent interpretation of the findings.

DESCRIPTOR(S)- *AMINO ACIDS; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *CIRCULAR DICHROISM; *EQUIPMENT; *FLUORIMETRY; *MOLECULAR STRUCTURE; *PROTEIN UNFOLDING MECHANISMS; *PROTEINS; *RM6 GENE; *ROP PROTEIN; *SPECTROSCOPIC TECHNIQUES; *SPEX FLUOROMAX FLUORIMETER; *TEMPERATURE EFFECTS; *TETRAMERIC LOOP DELETION MUTANT; *UNFOLDING REACTION

1998

Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination: ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA.

Gumbs, O. H.; Shaner, S. L.

Biochemistry, VOL. 37, NO. 33, 1998, PP. 11692-11706

The Escherichia coli RecA protein promotes DNA strand exchange in homologous recombination and recombinational DNA repair. Stopped-flow kinetics and fluorescence resonance energy transfer (FRET) were used to study RecA-mediated strand exchange between a 30-bp duplex DNA and a homologous single-stranded 50mer. In our standard assay, one end of the dsDNA helix was labeled at apposing 5' and 3' ends with hexachlorofluorescein and fluorescein, respectively. Strand exchange was monitored by the increase in fluorescence emission resulting upon displacement of the fluorescein-labeled strand from the initial duplex. The potential advantages of FRET in study of strand exchange are that it noninvasively measures real-time kinetics in the previously inaccessible millisecond time regime and offers great sensitivity. The oligonucleotide substrates model short-range mechanistic effects that might occur within a localized region of the ternary complex formed between RecA and long DNA molecules during strand exchange. Reactions in the presence of ATP with 0.1 mu-M duplex and 0.1-1.0 mu-M ss50mer showed triphasic kinetics in 600 s time courses, implying the existence of three mechanistic steps subsequent to presynaptic filament formation. The observed rate constants for the intermediate phase were independent of the concentration of ss50mer and most likely characterize a unimolecular isomerization of the ternary complex. The observed rate constants for the first and third phases decreased with increasing ss50mer concentration. Kinetic experiments performed with the nonhydrolyzable analogue ATP-gamma-S showed overall changes in fluorescence emission identical to those observed in the presence of ATP. In addition, the observed rate constants for the two fastest reaction phases were identical in ATP or ATP-gamma-S. The observed rate constant for the slowest phase showed a 4-fold reduction in the presence of ATP-gamma-S. Results in ATP-gamma-S using an alternate fluorophore labeling pattern suggest a third ternary intermediate may form prior to ssDNA product release. The existence of two or three ternary intermediates in strand exchange with a 30 bp duplex suggests the possibility that the step size for base pair switching may be 10-15 bp. Products of reactions in the presence of ATP and ATP-gamma-S, with and without proteinase K treatment, were analyzed on native polyacrylamide gels. In reactions in which only short-range Rec-A-DNA interactions were important, ATP hydrolysis was not required for recycling of RecA from both oligonucleotide products. Hydrolysis or deproteinization was required for RecA to release the heteroduplex product, but not the outgoing single strand.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ATP; *BIOCHEMISTRY AND BIOPHYSICS; *CHARACTERIZATION METHOD; *CHEMILUMINESCENT DETECTION; *DETECTION METHOD; *DETECTION-LABELING TECHNIQUES; *DNA; *EMISSION SPECTRA; *EQUIPMENT; *FLUORESCENCE RESONANCE ENERGY TRANSFER TECHNIQUE; *GEL ELECTROPHORESIS; *GENE MAPPING; *HI-TECH SCIENTIFIC SF-51 STOPPED FLOW APPARATUS; *HYDROLYSIS; *METHODOLOGY; *MOLECULAR PROBE TECHNIQUES; *POLYACRYLAMIDE GEL ELECTROPHORESIS; *PROTEIN-DNA INTERACTION; *RECA PROTEIN; *RECOMBINANT DNA TECHNOLOGY; *SEPARATION METHOD; *SOUTHERN BLOTTING; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROMAX ; *STOPPED FLOW KINETICS TECHNIQUE

1998

The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae.

Rossjohn, J.; Gilbert, R. J. C.; Crane, D.; Morgan, P. J.; Mitchell, T. J.; Rowe, A. J.; Andrew, P. W.; Paton, J. C.; Tweten, R. K.; Parker, M. W.

Journal of Molecular Biology, VOL. 284, NO. 2, 1998, PP. 449-461

Pneumolysin, a member of the thiol-activated cytolysin family of toxins, is a virulence factor from the Gram-positive bacterium Streptococcus pneumoniae. The toxin forms large oligomeric pores in cholesterol-containing membranes of eukaryotic cells. A plethora of biochemical and mutagenesis data have been published on pneumolysin, since its initial characterization in the 1930s. Here we present an homology model of the monomeric and oligomeric forms of pneumolysin based on the recently determined crystal structure of perfringolysin O and electron microscopy data. A feature of the model is a striking electronegative surface on parts of pneumolysin that may reflect its cytosolic location in the bacterial cell. The models provide a molecular basis for understanding the effects of published mutagenesis and biochemical modifications on the toxic activity of pneumolysin. In addition, spectroscopic data are presented that shed new light on pneumolysin activity and have guided us to hypothesise a detailed model of membrane insertion. These data show that the environment of some tryptophan residues changes on insertion and/or pore formation. In particular, spectroscopic analysis of a tryptophan mutant, W433F, suggests it is the residue mainly responsible for the observed effects. Furthermore, there is no change in the secondary structure content when the toxin inserts into membranes. Finally, the basis of the very low activity shown by a pneumolysin molecule from another strain of S. pneumoniae may be due to the movements of a key domain-domain interface. The molecular basis of pneumolysin-induced complement activation may be related to the structural similarity of one of the domains of pneumolysin to Fc, rather than the presumed homology of the toxin to C-reactive protein as previously suggested.

DESCRIPTOR(S)- *ACTIVITY ASSAYS; *ANALYSIS; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *C-REACTIVE PROTEIN; *CIRCULAR DICHROISM; *EQUIPMENT; *FLUORESCENCE SPECTROSCOPY; *HEMOLYTIC ACTIVITY ASSAY; *JASCO J-720 SPECTROPOLARIMETER; *METHODOLOGY; *PNEUMOLYSIN; *POLYACRYLAMIDE GEL ELECTROPHORESIS; *PURIFICATION; *SDS POLYACRYLAMIDE GEL ELECTROPHORESIS; *SDS-PAGE; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROMAX SINGLE-PHOTON-COUNTING SPECTROFLUORIMETER; *STREPTOCOCCUS PNEUMONIAE; *TOXIN; *VIRULENCE FACTOR

1998

The Cdc42-Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation.

Rudolph, M. G.; Bayer, P.; Abo, A.; Kuhlmann, J.; Vetter, I. R.; Wittinghofer, A.

Journal of Biological Chemistry, VOL. 273, NO. 29, 1998, PP. 18067-18076

Wiskott Aldrich syndrome is a rare hereditary disease that affects cell morphology and signal transduction in hematopoietic cells. Different size fragments of the Wiskott Aldrich syndrome protein, W4, W7 and W13, were expressed in Escherichia coli or obtained from proteolysis. All contain the GTPase binding domain (GBD), also called Cdc42/Rac interactive binding region (CRIB), found in many putative downstream effectors of Rac and Cdc42. We have developed assays to measure the binding interaction between these fragments and Cdc42 employing fluorescent N-methylanthraniloyl-guanine nucleotide analogues. The fragments bind with submicromolar affinities in a GTP-dependent manner, with the largest fragment having the highest affinity, showing that the GBD/CRIB motif is necessary but not sufficient for tight binding. Rate constants for the interaction with W13 have been determined via surface plasmon resonance, and the equilibrium dissociation constant obtained from their ratio agrees with the value obtained by fluorescence measurements. Far UV circular dichroism spectra show significant secondary structure only for W13, supported by fluorescence studies using intrinsic protein fluorescence and quenching by acrylamide. Proton and 15N NMR measurements show that the GBD/CRIB motif has no apparent secondary structure and that the region C-terminal to the GBD/CRIB region is alpha-helical. The binding of Cdc42 induces a structural rearrangement of residues in the GBD/CRIB motif, or alternatively, the Wiskott Aldrich syndrome protein fragments have an ensemble of conformations, one of which is stabilized by Cdc42 binding. Thus, in contrast to Ras effectors, which have no conserved sequence elements but a defined domain structure with ubiquitin topology, Rac/Cdc42 effectors have a highly conserved binding region but no defined domain structure in the absence of the GTP-binding protein. Deviating from common belief GBD/CRIB is neither a structural domain nor sufficient for tight binding as regions outside this motif are necessary for structure formation and tight interaction with Rho/Rac proteins.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES; *ANALYTICAL METHOD; *BIACORE AB; *BIACORE SYSTEM; *BIOCHEMISTRY AND BIOPHYSICS; *BRUKER; *BRUKER DRX-500 SPECTROMETER; *CDC42; *CDC42-NUCLEOTIDE COMPLEX PURIFICATION; *CDC42-RAC INTERACTIVE BINDING REGION; *CLONING TECHNIQUES; *CRIB; *ESCHERICHIA COLI; *EXPRESSION SYSTEM; *FAR UV CIRCULAR DICHROISM; *FLUORESCENT PROBE; * FLUOROMAX SPECTROFLUORIMETER; *GENETIC METHOD; *GTPASE BINDING DOMAIN; *IMAGING TECHNIQUES; *INTERACTIONS; *ISOLATION-PURIFICATION TECHNIQUES: CB; *JASCO; *J710 SPECTROPOLARIMETER; *LABORATORY EQUIPMENT; *METHODOLOGY; *N-METHYLANTHRANILOYL-GUANINE NUCLEOTIDE ANALOGUE; *NITROGEN-14 NMR; *PROTON NMR; *PURIFICATION METHOD; *RHO-RAC PROTEINS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX INDUSTRIES; *STRUCTURE FORMATION; *SURFACE PLASMON RESONANCE; *TIGHT BINDING; *WASP FRAGMENT CLONING; *WASP FRAGMENT PURIFICATION; *WISKOTT ALDRICH SYNDROME PROTEIN

1998

Tertiary structure-dependence of misfolding substitutions in loops of the maltose-binding protein.

Raffy, S.; Sassoon, N.; Hofnung, M.; Betton, J-M.

Protein Science, VOL. 7, NO. 10, 1998, PP. 2136-2142

We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the alpha-I/beta-B loop, of the N-domain that are critical for in vivo folding of the maltose-binding protein (MalE31). The tertiary context-dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent alpha-beta loop of the C-domain to the same amino acid substitutions (MalE219). Moving the loop mutation from the N- to the C-domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates in Escherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MaIE31 indicate that a competing off-pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of supersecondary structures of the N-domain is a rate-limiting step in the folding pathway of MalE.

DESCRIPTOR(S)- *ALPHA-BETA LOOP; *AMINO-TERMINAL DOMAIN; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BIOCHEMISTRY AND BIOPHYSICS; *ESCHERICHIA COLI; *EXPRESSION SYSTEM; *EXPRESSION VECTOR; * FLUOROMAX SPECTROFLUOROMETER; *FOLDING PATHWAY; *GEL ELECTROPHORESIS; *LABORATORY EQUIPMENT; *LIGHT-SCATTERING ANALYSIS; *MALTOSE-BINDING PROTEIN; *METHODOLOGY; *MISFOLDING SUBSTITUTIONS; *MOLECULAR GENETIC METHOD; *MUTAGENESIS; *MUTANT; *PROTEIN ENGINEERING; *P1H PLASMID; *SDS-PAGE; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SITE-DIRECTED MUTAGENESIS; *STABILITY; *STRAIN-POP6499; *STRAIN-POP6590; *STRUCTURE; *WILD-TYPE

1998

Structural transitions accompanying the activation of peptide binding to the endoplasmic reticulum Hsp90 chaperone GRP94.

Wearsch, P. A.; Voglino, L.; Nicchitta, C. V.

Biochemistry, VOL. 37, NO. 16, 1998, PP. 5709-5719

GRP94, the endoplasmic reticulum Hsp90 paralog, binds a diverse array of peptides, a subset of which are suitable for assembly onto nascent MHC class I molecules. At present, the mechanism, site, and regulation of peptide binding to GRP94 are unknown. Using VSVS, the immunodominant peptide epitope of the vesicular stomatitis virus, and native, purified GRP94, we have investigated GRP94-peptide complex formation. The formation of stable GRP94-VSV8 complexes was slow; competition studies demonstrated that peptide binding to GRP94 was specific. VSV8 binding to GRP94 was stimulated 2-fold or 4-fold, respectively, following chemical denaturation/renaturation or transient heat shock. The activation of GRP94-peptide binding occurred coincident with a stable, tertiary conformational change, as identified by tryptophan fluorescence and proteolysis studies. Analysis of GRP94 secondary structure by circular dichroism spectroscopy indicated an identical alpha-helical content for the native, chemically denatured/renatured, and heat-shocked forms of GRP94. Through use of the environment-sensitive fluorophores acrylodan and Nile Red, it was observed that the activation of peptide binding was accompanied by enhanced peptide and solvent accessibility to a hydrophobic binding site(s). Peptide binding to native or activated GRP94 was identical in the presence or absence of ATP or ADP. These results are discussed with respect to a model in which peptide binding to GRP94 occurs within a hydrophobic binding pocket whose accessibility is conformationally regulated in an adenine nucleotide-independent manner.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES; *ANALYTICAL GEL FILTRATION CHROMATOGRAPHY; *ANALYTICAL METHOD; *AVIV ASSOCIATES CIRCULAR DICHROISM SPECTROMETER MODEL 62DS; *BIOCHEMISTRY AND BIOPHYSICS; *CHROMATOGRAPHIC TECHNIQUES; *CIRCULAR DICHROISM SPECTROSCOPY; *ENDOPLASMIC RETICULUM; *EQUIPMENT; *FLUORESCENCE SPECTROSCOPY; * FLUOROMAX SPECTROFLUOROMETER; *GRP94; *HSP90 CHAPERONE; *METHODOLOGICAL APPROACH; *METHODSFINDER; *PEPTIDE BINDING; *PROTEIN PURIFICATION; *PURIFICATION METHOD; *SPECTROSCOPIC TECHNIQUES; *SPEX INDUSTRIES INCORPORATED; *STRUCTURE; *TSK-GEL G3000 SW COLUMN CHROMATOGRAPH

1998

Rapid assembly of Alzheimer-like paired helical filaments from microtubule-associated protein tau monitored by fluorescence in solution.

Friedhoff, P.; Schneider, A.; Mandelkow, E-M.; Mandelkow, E.

Biochemistry, VOL. 37, NO. 28, 1998, PP. 10223-10230

Alzheimer's disease is characterized by the progressive deposition of two types of fibers in the affected brains, the amyloid fibers (consisting of the A-beta peptide, generating the amyloid plaques) and paired helical filaments (PHFs, made up of tau protein, forming the neurofibrillary tangles). While the principles of amyloid aggregation are known in some detail, the investigation of PHF assembly has been hampered by the low efficiency of tau aggregation, the requirement of high protein concentrations, and the lack of suitable detection methods. Here we report a quantitative assay system that permits monitoring of the assembly of PHFs in real time by the fluorescence of dyes such as thioflavine S or T. Using this assay, we evaluated parameters that influence the efficiency of filament formation. Disulfide-linked dimers of tau constructs representing the repeat domain assemble into PHFs most efficiently, but other tau isoforms or constructs form bona fide PHFs as well. The rate of assembly is greatly enhanced by polyanions such as RNA, heparin, and notably polyglutamate which resembles the acidic tail of tubulin. The assembly is optimal at pH apprx 6 and low ionic strengths ( lt 50 mM) and increases steeply with temperatures above 30 degree C, indicating that it is an entropy-driven process.

DESCRIPTOR(S)- *ALZHEIMER-LIKE PAIRED HELICAL FILAMENTS; *ANALYSIS; *ANALYTICAL METHOD; *ASSEMBLY MONITORING; *BIOCHEMISTRY AND BIOPHYSICS; *ELECTRON MICROSCOPY; *FLUORESCENCE SPECTROSCOPY; *LABORATORY EQUIPMENT; *METHODOLOGY; *MICROSCOPY: CB; *MICROTUBULE-ASSOCIATED PROTEIN TAU; *RAPID ASSEMBLY IN SOLUTION; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX FLUOROMAX INSTRUMENT; *SPEX INSTRUMENTS SA; *VISUALIZATION METHOD

1998

Protoporphyrin IX Fluorescence Induced in Basal Cell Carcinoma by Oral Aminolevulinic Acid.

Tope, W., Ross, V., Kollias, N. Martin, A., Gillies, R., and Anderson, R. R.;

Photochemistry and Photobiology, Volume 67(2), (1998), pp. 249 - 255.

 

1998

Phage P22 tailspike protein: Removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

Miller, S.; Schuler, B.; Seckler, R.

Protein Science, VOL. 7, NO. 10, 1998, PP. 2223-2232

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the aminoterminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degree C occurs at a rate of 9.2 times 10-4 S-1. It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.

DESCRIPTOR(S)- *AMICON; *ANALYSIS; *ANALYTICAL METHOD; *ANION EXCHANGE CHROMATOGRAPHY; *AVIV 62A-DS SPECTROPOLARIMETER; *BACTERIOPHAGE P22; *CARY 1 SPECTROPHOTOMETER; *CENTRICON 30 MICROCONCENTRATOR; *CIRCULAR DICHROISM; *CLONING METHOD; *COLUMN CHROMATOGRAPHY; *CRYSTALLIZATION TECHNIQUES; *DNA AMPLIFICATION; *ENZYMOLOGY; *ESCHERICHIA COLI; *EXPRESSION SYSTEM; *FILTRATION TECHNIQUES; *FLUORESCENCE SPECTROSCOPY; *FOLDING KINETICS; *FOLDING MUTATIONS; *GEL ELECTROPHORESIS; *GEL FILTRATION; *HANGING DROP CRYSTALLIZATION; *HEAD-BINDING DOMAIN; *JASCO; *JASCO 715 SPECTROPOLARIMETER; *LABORATORY EQUIPMENT; *METHODOLOGY; *MOLECULAR GENETIC METHOD; *MUTAGENESIS; *NATIVE STATE; *PARALLEL BETA HELIX; *PCR; *PHAGE P22 TAILSPIKE ENDORHAMNOSIDASE; *POLYMERASE CHAIN REACTION; *PROTEIN ENGINEERING; *PROTEIN FOLDING INTERMEDIATES; *PURIFICATION METHOD; *SDS-PAGE; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SECOND-SITE SUPPRESSOR MUTATIONS; *SILVER STAINING; *SITE-DIRECTED MUTAGENESIS; *SPECTROSCOPIC TECHNIQUES: CB; *SPECTROSCOPY: CB; *SPEX FLUOROMAX ; *STAINING-VISUALIZATION; *STRAIN-JM83; *THERMA STABILITY; *VARIAN

1998

Monitoring the sizes of denatured ensembles of staphylococcal nuclease proteins: Implications regarding m values, intermediates, and thermodynamics.

Baskakov, I. V.; Bolen, D. W.

Biochemistry, VOL. 37, NO. 51, 1998, PP. 18010-18017

Fluorescence and size-exclusion chromatography (SEC) are used to monitor urea denaturation of wild-type staphylococcal nuclease (SN) as well as the m+ and m- mutants A69T and V66W, respectively. It is found that the SEC partition coefficient, 1/K-d, is directly proportional to the Stokes radii of proteins. From the Stokes radii, the denatured ensembles of the three proteins are found to be highly compact in the limit of low urea concentration and expand significantly with increasing urea concentration. The m values from fluorescence-detected denaturation of the SN proteins are generally considered to reflect the relative sizes of denatured ensembles. However, the rank order of m values of the SN proteins studied do not correspond to the rank order of denatured ensemble sizes detected by 1/K-d, suggesting that m values reflect more than just surface area increases on denaturation. SEC provides two complementary ways to demonstrate the existence of intermediates in urea denaturation and illustrates that V66W undergoes a three-state transition. Fluorescence-detected urea denaturations of A69T and wt SN do not correspond with 1/K-d-detected denaturation profiles, a result that would ordinarily mean that the transitions are non-two-state. However, this interpretation fails to recognize the rapidly changing size and thermodynamic character of the denatured ensembles of these proteins both within and outside of the transition zone. The implications of the changing sizes and thermodynamic character of the denatured ensembles for SN proteins are manifold, requiring a reconsideration of the thermodynamics of proteins whose denatured ensembles behave as those of SN proteins.

DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *CHROMATOGRAPHIC TECHNIQUES; *DENATURATION; *ELECTROPHORETIC TECHNIQUES; *ENZYMOLOGY; *EQUIPMENT; *M VALUES; *MOLECULAR CHARACTERISTICS; *MOLECULAR INTERMEDIATES; *MOLECULAR SIZED; *NUCLEASES; *PHENOMENEX; *PHENOMENEX BIOSEP SEC-S3000 HPLC SYSTEM; *PROTEIN DENATURATION; *PROTEIN FOLDING MECHANISMS; *PROTEINS; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SIZE-EXCLUSION CHROMATOGRAPHY; *SPECTROFLUORIMETRY; *SPEX; *SPEX FLUOROMAX SPECTROFLUORIMETER; *STAPHYLOCOCCAL NUCLEASE PROTEINS; *STAPHYLOCOCCI; *THERMODYNAMICS

1998

Flexibility of helix 2 in the human glutathione transferase P1-1. Time-resolved fluorescence spectroscopy.

Stella, L.; Caccuri, A. M.; Rosato, N.; Nicotra, M.; Lo, Bello M.; De Matteis, F.; Mazzetti, A. P.; Federici, G.; Ricci, G.

Journal of Biological Chemistry, VOL. 273, NO. 36, 1998, PP. 23267-23273

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.

DESCRIPTOR(S)- *ANALYSIS; *ANALYTICAL METHOD; *EC 2.5.1.18; *ENZYMOLOGY; *ESCHERICHIA COLI; *EXPRESSION SYSTEM; *HELIX 2 FLEXIBILITY; *HUMAN GLUTATHIONE TRANSFERASE P1-1; *LABORATORY EQUIPMENT; *METHODOLOGY; *MOLECULAR GENETIC METHOD; *MUTAGENESIS; *NEODYMIUM-YAG LASER; *PROTEIN ENGINEERING; *SITE-DIRECTED MUTAGENESIS; *SPECTROSCOPY: CB; *SPEX; *SPEX FLUOROMAX PHOTON COUNTING SPECTROFLUOROMETER; *TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

1998

Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitolyphosphatidylcholine bilayers to phospholipase A-2.

Henshaw, J. B.; Olsen, C. A.; Farnbach, A. R.; Nielson, K. H.; Bell, J. D.

Biochemistry, VOL. 37, NO. 30, 1998, PP. 10709-10721

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A-2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A-2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A-2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, unionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.

DESCRIPTOR(S)- *AGKISTRODON-PISCIVORUS-PISCIVORUS VENOM PHOSPHOLIPASE A-2; *ANALYSIS; *ANALYTICAL METHOD; *AVANTI POLAR LIPIDS; *BIOCHEMISTRY AND BIOPHYSICS; *CATALYTIC SUSCEPTIBILITY; *DIPALMITOLYPHOSPHATIDYLCHOLINE BILAYERS; * FLUOROMAX SPECTROFLUOROMETER; *GREG PC PHOTON-COUNTING SPECTROFLUOROMETER; *ISS; *LABORATORY EQUIPMENT; *LYSOLECITHIN; *METHODOLOGY; *PALMITIC ACID; *PC-1 PHOTON-COUNTING SPECTROFLUOROMETER; *SIGMA; *SPECTROFLUORIMETRY; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX INDUSTRIES

1998

Correlation of fluorescence and circular dichroism spectroscopy with electrospray ionization mass spectrometry in the determination of tertiary conformational changes in calcium-binding proteins.

Veenstra, T. D.; Johnson, K. L.; Tomlinson, A. J.; Kumar, R.; Naylor, S.

Rapid Communications in Mass Spectrometry, VOL. 12, NO. 10, 1998, PP. 613-619

We compared changes in the fluorescence, circular dichroism (CD) and multiply charged electrospray ionization mass spectrometry (ESI-MS) spectra of three calcium (Ca-2+)-binding proteins upon the binding of Ca+. The proteins used were rat brain calbindin D-28K and two deletion mutants, one lacking EF-hand 2 (calbindin DELTA-2) and the other lacking EF-hands 2 and 6 (calbindin DELTA-2,6). Large changes in the intrinsic protein fluorescence spectrum were seen upon the addition of Ca-2+ to calbindin D-28K and DELTA-2, while a less significant change was observed for calbindin DELTA-2,6. In a fluorescent study in which p-toluidinyl-2-naphthalene-6-sulfonate, a fluorescent probe which binds to hydrophobic surfaces within proteins, was used; calbindin D-28K and DELTA-2 again showed a greater change in fluorescence intensity upon Ca-2+-binding than calbindin DELTA-2,6. Near UV-CD studies, which measure changes within the tertiary structure of a protein, showed greater changes in the spectrum of calbindin D-28K and DELTA-2 compared to calbindin DELTA-2,6 upon Ca-2+binding. Far UV-CD studies, which measures changes within the secondary structure of a protein, however, showed that the spectrum of all three proteins underwent only minor changes upon metal-binding. The ESIMS studies showed that as the proteins were titrated with Ca-2+ a gradual shift in the mass envelope from higher to lower charge states occurs. In the case of calbindin D-28K and calbindin DELTA-2, however, a complete shift in the mass envelope towards the lower charge states is observed upon saturation with Ca-2+, whereas for calbindin DELTA-2,6, the shift in the charge states is still relatively evenly distributed between high and low charge states. Changes within the ESI-MS spectrum observed upon the addition of Ca-2+ correlated with Ca-2+-induced changes observed with near-ultraviolet CD, intrinsic fluorescence spectroscopy, and spectroscopy using the fluorescent probe. Changes in the far ultraviolet-CD spectra of the calbindins, however, did not correlate with changes in the ESI-MS spectra upon calcium binding. The results show that ESI-MS can be use to detect changes in the tertiary structure of calcium-binding proteins induced by the binding of metal to the proteins.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES; *BIOCHEMISTRY AND BIOPHYSICS; *CALBINDIN; *CALCIUM-BINDING PROTEINS; *CHARACTERIZATION; *CHARACTERIZATION METHOD; *CIRCULAR DICHROISM SPECTROSCOPY; *ELECTROSPRAY IONIZATION MASS SPECTROMETRY; *EQUIPMENT; *FINNIGAN MAT 900 MASS SPECTROMETER; *FLUORESCENCE SPECTROSCOPY; * FLUOROMAX FLUORESCENCE SPECTROMETER; *JASCO J-710 SPECTROPOLARIMETER; *METHODOLOGY; *METHODSFINDER; *SPECTROSCOPIC TECHNIQUES; *SPEX INSTRUMENTS; *TERTIARY CONFORMATIONAL CHANGES

1998

Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE.

Pierpaoli, E. V., Sandmeier, E., Schoenfeld, H-J., and Christen, P.;

Journal of Biological Chemistry, Volume 273, Number 12, (1998), pp. 6643 - 6649.

 

1998

Calcium-dependent membrane penetration is a hallmark of the C2 domain of cytosolic phospholipase A2 whereas the C2A domain of synaptotagmin binds membranes electrostatically.

Davletov, B.; Perisic, O.; Williams, R. L.

Journal of Biological Chemistry, VOL. 273, NO. 30, 1998, PP. 19093-19096

C2 domains have been identified in a wide range of intracellular proteins, including lipid modifying enzymes, protein kinase, GTPases, and proteins involved in membrane trafficking. Many C2 domains bind membranes in a calcium-dependent manner. The first C2 domain from synaptotagmin I (SytIC2A) and the C2 domain from cytosolic phospholipase A2 (cPLA2C2) are among the best characterized C2 domains in terms of their structures and calcium binding. Here we demonstrate that the protein lipid interaction is dramatically different for these two domains. Photolabeling with 3-(trifluoromethyl)-3-(m-(125I)iodophenyl)diazirine ((125I)TID) in the presence of phospholipid vesicles indicates that cPLA2C2 penetrates into the hydrophobic region of the membrane. Hydrophobic surfaces on cPLA2C2 are exposed even in the absence of calcium, but only in its presence does the domain penetrate into the nonpolar core of the membrane. The interaction of SytIC2A with phospholipid membranes is primarily electrostatic with binding being abolished in 500 mM NaCl. Because soluble phospholipid head group analogues do not compete with binding of either SytIC2A or cPLA2C2 to vesicles, it is likely that membrane binding by these domains involves multiple interactions.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *CALCIUM-DEPENDENT MEMBRANE PENETRATION; *CYTOSOLIC PHOSPHOLIPASE A2; *C2 DOMAIN; *C2A DOMAIN; *DETECTION-LABELING TECHNIQUES; *ELECTROSTATIC MEMBRANE BINDING; *ENZYMOLOGY; *EXPRESSION; *FLUORESCENCE ANALYSIS; * FLUOROMAX-2 FLUORIMETER; *GEL ELECTROPHORESIS; *HYDROPHOBIC SURFACES; *ISOLATION-PURIFICATION TECHNIQUES: CB; *JOBIN YVON-SPEX; *LABORATORY EQUIPMENT; *LARGE MULTILAMELLAR VESICLES; *MEMBRANES; *METHODOLOGY; *MOLECULAR PROBES; *PHASE SEPARATION; *PROTEIN RADIOLABELING; *PROTEIN-LIPID INTERACTION; *PURIFICATION; *PURIFICATION METHOD; *SDS-PAGE; *SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS; *SEPARATION METHOD; *SIGMA; *SYNAPTOTAGMIN; *TRITON X-114; *4,4'-DIANILINO-1,1'-BINAPHTHYL-5,5'-DISULFONIC ACID

1998

Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli.

Chinchilla, D.; Schwarz, F. P.; Eisenstein, E.

Journal of Biological Chemistry, VOL. 273, NO. 36, 1998, PP. 23219-23224

Shifts in the sigmoidal kinetics of allosteric threonine deaminase promoted by isoleucine and valine binding control branched chain amino acid biosynthesis in Escherichia coli. A highly conserved alpha-helix in the C-terminal regulatory domain of the tetrameric enzyme was previously implicated in effector binding and feedback inhibition. Double (447, 451) and triple (447, 451, 454) alanine replacements for the conserved amino acids leucine 447, leucine 451, and leucine 454 in this region yield enzyme variants that show increased sigmoidality in steady-state kinetics, and which are less sensitive to the allosteric modifiers isoleucine and valine. Equilibrium binding studies using fluorescence, enzyme kinetic, and calorimetric approaches indicate that the enzyme variants possess reduced affinity for isoleucine and valine, and suggest that heterotropic ligands can bind to the same site to promote their different effects. The increase in sigmoidal kinetics for the mutants relative to wild-type threonine deaminase may be attributable to the elimination of L-threonine binding to the effector sites, which activates the wild-type enzyme. Enzyme kinetic data and isotherms for active site ligand binding to the mutants can be analyzed in terms of a simple two-state model to yield values for allosteric parameters that are consistent with previous estimates based on an expanded two-state model for homotropic cooperativity for threonine deaminase.

DESCRIPTOR(S)- *ACTIVE SITE LIGAND BINDING; *ALLOSTERIC EFFECTOR BINDING; *AMINO ACID SUBSTITUTIONS; *ANALYSIS; *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *BECKMAN OPTIMA XL-A ANALYTICAL ULTRACENTRIFUGE; *CARBOXY-TERMINAL REGULATORY DOMAIN; *ENZYMOLOGY; *ESCHERICHIA COLI; *FLUORESCENCE SPECTROSCOPY; * FLUOROMAX 2000 SPECTROFLUORIMETER; *HOMOTROPIC COOPERATIVITY; *LABORATORY EQUIPMENT; *METHODOLOGY; *MICROCAL OMEGA TITRATION CALORIMETER; *MOLECULAR GENETIC METHOD; *MUTAGENESIS; *MUTANT; *MUTATION; *PROTEIN ENGINEERING; *SEDIMENTATION EQUILIBRIUM ANALYSIS; *SITE-DIRECTED MUTAGENESIS; *SPECTROSCOPIC TECHNIQUES: CB; *SPEX INDUSTRIES; *STEADY STATE KINETICS; *THREONINE DEAMINASE; *TITRATION CALORIMETRY

1998

A method for studying plasma membrane transport with intact cells using computerized fluorometry.

Wielinga, P. R.; Heijn, M.; Westerhoff, H.; Lankelma, J.

Analytical Biochemistry, VOL. 263, NO. 2, 1998, PP. 221-231

A new method is presented for measuring rapid efflux of fluorescent compounds from monolayer cells. Cells grown on a glass coverslip were loaded with a fluorescent substrate. Thereafter, the coverslip was installed outside the light path in a stirred and thermostated cuvette of a fluorometer. The efflux was recorded by measuring the changes of fluorescence in the extracellular medium. The method was used to study the kinetics of active and passive plasma membrane transport of the P-glycoprotein substrates rhodamine 123 and daunorubicin. The method has advantages over other methods: (1) no radioactively labeled substrate is needed, (2) fluorescence of the transported substrate is not compromised by the cells, (3) changes in the extracellular concentration of the substrate can be monitored continuously and therefore a substantial improvement of the kinetic resolution is obtained, and (4) the measurement setup is relatively simple and a standard fluorometer can be used. From the efflux data, cellular transport parameters could be calculated, such as passive permeation coefficients and active transport rates.

DESCRIPTOR(S)- *ANALYTICAL METHOD; *COMPUTERIZED FLUOROMETRY; *DAUNORUBICIN; * FLUOROMAX SPECTROFLUOROMETER; *INTACT CELLS; *KB-V1 CELL LINE; *KB-3-1 CELL LINE; *LABORATORY EQUIPMENT; *MEMBRANES; *METHODOLOGY; *P-GLYCOPROTEIN SUBSTRATE; *PHOTOMETRY: CT; *PLASMA MEMBRANE TRANSPORT; *RHODAMINE 123; *SIGMA; *SPECIA; *SPEX INDUSTRIES; *TRANSPORT

1998

2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: Metal-ion and base sequence effects.

Stivers, J. T.

Nucleic Acids Research, VOL. 26, NO. 16, 1998, PP. 3837-3844

Metal-ion and sequence dependent changes in the stacking interactions of bases surrounding abasic (AB) sites in 10 different DNA duplexes were examined by incorporating the fluorescent nucleotide probe 2-aminopurine (2-AP), opposite to the site (AB-AP-opp) or adjacent to the site (AB-Ap-adj) on either strand. A detailed study of the fluorescence emission and excitation spectra of these AB duplexes and their corresponding parent duplexes indicates that AB-AP-opp is significantly less stacked than 2-AP in the corresponding normal duplex. In general, AB-Ap-adj on the AB strand is stacked, but AB-Ap-adj on the opposite strand shows destabilized stacking interactions. The results also indicate that divalent cation binding to the AB duplexes contributes to destabilizaton of the base stacking interactions of AB-AP-opp, but has little or no effect on the stacking interactions of AB-AP-adj. Consistent with these results, the fluorescence of AB-AP-opp is 18-30-fold more sensitive to an externally added quenching agent than the parent normal duplex. When uracil DNA glycosylase binds to AB-AP-opp in the presence of 2.5 mM MgCl-2, a 3-fold decrease in fluorescence is observed (K-d = 400 +- 90 nM) indicating that the unstacked 2-AP-opp becomes more stacked upon binding. On the basis of these fluorescence studies a model for the local base stacking interactions at these AB sites is proposed.

DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION TECHNIQUES: CB; *ANALYTICAL METHOD; *APPLIED BIOSYSTEMS; *APPLIED BIOSYSTEMS 390 SYNTHESIZER; *BASE SEQUENCE EFFECTS; *BASE STACKING INTERACTIONS; *CONTINUOUS FLUORESCENCE KINETIC ASSAY; *DETECTION METHOD; *DETECTION-LABELING TECHNIQUES; *DNA; *ESCHERICHIA COLI; *FLUORESCENCE EMISSION SPECTROSCOPY; *FLUORESCENT NUCLEOTIDE PROBE; *FLUOROPHOTOMETRY: CB; *GEL ELECTROPHORESIS; *LABORATORY EQUIPMENT; *LIQUID CHROMATOGRAPHY; *METAL-ION EFFECTS; *METHODOLOGY; *MOLECULAR GENETICS; *OLIGODEOXYNUCLEOTIDE SYNTHESIS; *POLYACRYLAMIDE GEL ELECTROPHORESIS; *PURIFICATION METHOD; *REVERSE-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; *REVERSE-PHASE HPLC; *SPEX FLUOROMAX SPECTROFLUOROMETER; *SYNTHESIS-MODIFICATION TECHNIQUES; *SYNTHETIC METHOD; *TITRATION; *URACIL DNA GLYCOSYLASE; *2-AMINOPURINE; *2-AMINOPURINE FLUORESCENCE STUDIES