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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2000

The ATT strand of AATcntdotATT trinucleotide repeats adopts stable hairpin structures induced by minor groove binding ligands.

Trotta, Edoardo; Del Grosso, Nicoletta; Erba, Maura; Paci, Maurizio

Biochemistry, VOL. 39, NO. 23, June 13, 2000, PP. 6799-6808

AATcntdotATT is the most abundant and also the most frequently polymorphic class of trinucleotide repeats in the human genome. To characterize its structural properties and conformational changes induced by minor groove ligands, (AAT)6 and (ATT)6 oligomers as well as their complexes with DAPI were investigated by electrophoretic mobility and UV thermal stability as well as fluorescence and NMR spectroscopy. The results show that individual (AAT)6 and (ATT)6 strands exist principally as monomeric non-hydrogen-bonded structures. Their individual interaction with DAPI induces the formation of base-paired structures with different thermal stabilities by quite spectroscopically distinct binding mechanisms. In the presence of DAPI, (ATT)6 forms a monomeric hairpin structure stabilized by two ligands located in the minor groove with a strong apparent binding constant of 3.4 X 106 M-1. The DAPI-induced (ATT)6 hairpin is characterized by well-stacked AcntdotT Watson-Crick and TcntdotT wobble base pairs, a high electrophoretic mobility, and a melting temperature of 41 degreeC. Interaction of DAPI with the complementary (AAT)6 strand favors less stable base-paired structures, and the results are consistent with electrostatic and hydrogen-bond interactions of the ligand with the phosphodiester backbone of (AAT)6 by minor involvement of DNA bases.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *groove binding ligands; *AAT-AAT trinucleotide repeats --hairpin structure; *4',6-diamidino-2-phenylindole; *electrophoresis --analytical method; *electrophoresis --electrophoretic techniques; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *molecular modeling --analytical method; *molecular modeling --Molecular Biology Techniques and Chemical Characterization; *Bruker AM 400 spectrometer --equipment; *Bruker AM 400 spectrometer --Bruker; *H-P model 8452A spectrophotometer --equipment; *H-P model 8452A spectrophotometer --Hewlett-Packard; *NMR spectroscopy --analytical method; *NMR spectroscopy --spectroscopic techniques: CB; *SPEX FluoroMax photon counting spectrofluorometer --equipment; *SPEX FluoroMax photon counting spectrofluorometer --SPEX

2000

Structure and dynamics of an acid-denatured protein G mutant.

Orban, John .; Alexander, Patrick; Bryan, Philip N.; Sari, Nese

Biochemistry, VOL. 39, NO. 5, Feb. 8,. 2000, PP. 965-977

NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of GB1 which is on the margin of stability. At 5 degreeC, F30H-GB1 is greater than 95% folded at pH 7.0 and is greater than 95% unfolded at pH 4.0. This range of stability is useful because the denatured state can be examined under relatively mild conditions which are optimal for folding GB1. We have assigned almost all backbone 15N, HN, and Halpha resonances in the acid-denatured state. Chemical shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild conditions than in the presence of chemical denaturants. The acid-denatured state populates nativelike conformations with both alpha-helical and beta-hairpin characteristics. To our knowledge, this is the first example of a denatured state with NOE and coupling constant evidence for beta-hairpin character. A number of non-native turn structures are also detected, particularly in the region corresponding to the beta1-beta2 hairpin of the folded state. Steady-state (1H-15N) NOE results demonstrate restricted backbone flexibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the beta3-beta4 hairpin, and the beta1-beta2 turn may serve as potential initiation sites for folding of GB. Furthermore, residual structure in acid-denatured F30H-GB1 is more extensive than in peptide fragments corresponding to the beta1-beta2, alpha-helix, and beta3-beta4 regions, suggesting additional medium-to-long-range interactions in the full-length polypeptide chain..

DESCRIPTOR(S)- *Methods and Techniques.; *Molecular Genetics (Biochemistry and Molecular Biophysics); *acid-denatured protein G.; *circular dichroism; *cloning; *kinetic analysis; *site-directed mutagenesis.; *Bruker DRX-500 spectrometer; *Bruker DRX-600 spectrometer; *DNA sequencing; *Jasco model J-720 spectropolarimeter; *KinTek Stopped-Flow model SF2001; *NMR spectroscopy; *NOESY nuclear Overhauser effect spectroscopy ; *SPEX FluoroMax spectrofluorimeter; *TOCSY total correlation spectroscopy ; *gene mutation; *protein conformation; *protein denaturation; *protein dynamics; *protein folding; *protein structure.

2000

Simultaneous registration of contraction and cytosolic calcium (ÝCa/sup 2+/¨/sub i/) of smooth muscle strips using front-surface fluorimetry

Ferreira, A. T.; Neri, R.; Oshiro, M. E. M.; Kanaide, H.

Journal of Fluorescence, J. Fluoresc. (USA), VOL. 10, NO. 3, Sept. 2000, PP. 223-8

Using front-surface fluorimetry with fura-2-loaded smooth muscle strips, simultaneous registration of the cytosolic calcium concentration (ÝCa/sup 2+/¨/sub i/) changes and tension development was done under the action of 40 mM KCl and the myotropic peptide 10/sup -6/ M angiotensin II. The strips were mounted vertically, connected to a force transducer that keeps a basal isometric tension of 0.5 g, and maintained in a bathing solution oxygenated at 37 degrees C. The fiber-optic platform FluoroMax-2 accessory 1950F was used to do the remote sensing for the samples. Light from the excitation spectrometer ( FluoroMax-2 ), alternating between 340 and 380 nm, was focused onto the fiber-optic bundle and directed to the smooth muscle strip. The fluorescence (505 nm) was collected and redirected to the emission port of the fluorimeter FluoroMax-2 The ratiometric method (R340/380) was used as an index of ÝCa/sup 2+/¨; change during smooth muscle contraction. All data, R340/380 and tension, were recorded using a computerized data acquisition system: Soft and Solution and GRAMS/386 of Galatic Industries Corporation.

DESCRIPTOR(S)- calcium; muscle; spectrochemical analysis

2000

Simultaneous registration of contraction and cytosolic calcium ( left bracket CaSUP2SUP plus right bracket SUBi) of smooth muscle strips using front-surface fluorimetry

Ferreira, Alice T.; Neri, Rogerio; Oshiro, Maria E.M.; Kanaide, Hideo

Journal of Fluorescence v 10 n 3 Sep 2000. p 223-228 2000

Using front-surface fluorimetry with fura-2-loaded smooth muscle strips, simultaneous registration of the cytosolic calcium concentration ( left bracket CaSUP2SUP plus right bracket SUBi) changes and tension development was done under the action of 40 mM KCl and the myotropic peptide 10SUP minus SUP6M angiotensin II. The strips were mounted vertically, connected to a force transducer that keeps a basal isometric tension of 0.5 g, and maintained in a bathing solution oxygenated at 37 degree C. The fiber-optic platform FluoroMax-2 accessory 1950F was used to do the remote sensing for the samples. Light from the excitation spectrometer ( FluoroMax-2 ), alternating between 340 and 380 nm, was focused onto the fiber-optic bundle and directed to the smooth muscle strip. The fluorescence (505 nm) was collected and redirected to the emission port of the fluorimeter FluoroMax-2 The ratiometric method (R340/380) was used as an index of left bracket CaSUP2SUP plus right bracket SUBi change during smooth muscle contraction. All data, R340/380 and tension, were recorded using a computerized data acquisition system: Soft & Solution and GRAMS/386 of Galatic Industries Corporation. (Author abstract) 9 Refs.

DESCRIPTOR(S)- Calcium; Data acquisition; Emission spectroscopy; Fiber optics; Fluorescence; Muscle; Photomultipliers; Positive ions; Potassium compounds

2000

PHOSPHATIDYLCHOLINE-FUNCTIONAL SURFACES VIA SEQUENTIAL GRAFTING REACTION

Pollack, S. K.; Bullen, Y. S.

2000, Washington, D.C., 20th-24th Aug.2000, p.1256-7

Successive hydrosilations are performed to create the desired biocompatible membrane. A silane derivative 5-hexenyltrichlorosilane is reacted to a Si/SiO2 surface to give a vinyl surface. With the aid of a platinum catalyst, this surface is then reacted with a 25-30% dimethylsiloxane-methylhydrosiloxane copolymer via a hydrosilation reaction. These layers are characterised by contact angle using a contact angle goniometer and attenuated total reflection by use of a Si/SiO2 prism. The final step of this process is to covalently bond phosphatidylcholine (PC) on to the surface via a hydrosilation reaction. This process cannot be easily followed; hence, a model is developed to follow the final attachment to the surface. With the aid of a platinum catalyst, a pyrene derivative is grafted on to the reactive surface by a hydrosilation reaction. The pyrene-treated glass is monitored by fluorescence spectroscopy using a SPEX FluoroMax-2 Attachment is confirmed, therefore, the PC is being attached and further studies, such as protein adsorption, would be performed. 6 refs.
NON POLYMER- PLATINUM

DESCRIPTOR(S)- ATTENUATED TOTAL REFLECTANCE SPECTROSCOPY; ATTENUATED TOTAL REFLECTION SPECTROSCOPY; BIOMEMBRANE; CATALYST; CHEMICAL MODIFICATION; CONTACT ANGLE; DATA; DIMETHYL SILOXANE COPOLYMER; FLUORESCENCE SPECTROSCOPY; GRAPH; HYDROSILATION; INSTITUTION; METHYLHYDROSILOXANE COPOLYMER; MODEL; PLASTIC; POLYMERISATION CATALYST; POLYMERISATION CATALYSTS; POLYMERIZATION CATALYST; PROTEIN ADSORPTION; SILICON COPOLYMER; SILICON-CONTAINING COPOLYMER; SILICONE COPOLYMER; SILOXANE COPOLYMER; SPECTROSCOPY; SURFACE TREATMENT; TABLES; TECHNICAL; THERMOPLASTIC; THERMOSET

2000

Parallel self-associated structures formed by T,C-rich sequences at acidic pH.

Taillandier, Eliane; Alexeev, Yakov I.; Brevnov, Maxim G.; Geinguenaud, Frederic; Gromova, Elizaveta S.; Liquier, Jean; Petrauskene, Olga V.

Biochemistry, VOL. 39, NO. 41, October 17, 2000, PP. 12650-12658

Oligonucleotides of nonregular heteropyrimidine sequences incorporating or not incorporating purine residues 5'-d(ACTCCCTTCTCCTCTCTA), 5'-d(ACTCCCTGGTCCTCTCTA), 5'-d(TCTCTCCTGGTCCCTCC), and 5'-d(TCTCTCCTCTTCCCTCC) can form self-associated parallel-stranded (ps) structures at pH 4-5.5. The ps structures were identified by studying at neutral and acidic pH UV melting transitions, FTIR spectra, and fluorescence of pyrene-labeled oligonucleotides as well as by chemical joining of 5'-phosphorylated oligonucleotides. A gel electrophoresis run for oligonucleotides 5'-d(TCTCTCCTCTTCCCTCC) and 5'-d(ACTCCCTTCTCCTCTCTA) has shown the formation of homoduplexes at low DNA strand concentrations. Ps structures are held by C-C+ base pairs and have N- and S-types of sugar puckering as detected by FTIR spectroscopy in the millimolar concentration range. Guanine inserts as well as thymine and purine inserts into an oligomeric cytosine sequence make the formation of the tetraplex i-motif unfavorable. MvaI restriction endonuclease, which recognizes the CCT/AGG sequence in DNA, does not cleave parallel pseudosubstrates.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *oligonucleotides --acidic pH; *oligonucleotides --analysis; *oligonucleotides --parallel self-associated structures; *oligonucleotides --T-C rich sequences; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *Fourier transform IR spectroscopy FTIR spectroscopy --analytical method; *Fourier transform IR spectroscopy FTIR spectroscopy --IR spectrophotometry: CB; *Perkin-Elmer Series 200 spectrophotometer --equipment; *Perkin-Elmer Series 200 spectrophotometer --Perkin-Elmer; *Spex FluoroMax spectrofluorometer --equipment; *DNA-protein interaction

2000

Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy.

Marino, John P.; Lacourciere, Karen A.; Stivers, James T.

Biochemistry, VOL. 39, NO. 19, May 16, 2000, PP. 5630-5641

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env, gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (KD = 20 +- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (KD = 0.24 +- 0.040 muM), the second site inhibited Rev binding in a competitive fashion (KD = 1.8 +- 0.8 muM), and the third much weaker site (or sites) is attributed to nonspecific binding (KD gtoreq 40 muM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *HIV-1 human immunodeficiency virus 1 (Retroviridae); *Animal Viruses; *Microorganisms; *Viruses; *Rev responsive element --analysis; *Rev responsive element --neomycin binding; *Rev responsive element --Rev peptide binding; *2-aminopurine 2'-O-methylriboside --fluorescent probe; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *Bruker AVANCE 500 spectrometer --equipment; *Bruker AVANCE 500 spectrometer --Bruker Instruments; *Bruker AVANCE 600 spectrometer --equipment; *Bruker AVANCE 600 spectrometer --Bruker Instruments; *NMR spectroscopy --analytical method; *NMR spectroscopy --spectroscopic techniques: CB; *SPEX Fluoromax-2 spectrofluorometer --equipment; *SPEX Fluoromax-2 spectrofluorometer --Instruments SA

2000

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. II. Ligand-mediated activation of GRP94 molecular chaperone and peptide binding activity.

Nicchitta, Christopher V.; Reed, Robyn C.; Wassenberg, James J.

Journal of Biological Chemistry, VOL. 275, NO. 30, July 28, 2000, PP. 22806-22814

The N-terminal domain of eukaryotic Hsp90 proteins contains a conserved adenosine nucleotide binding pocket that also serves as the binding site for the Hsp90 inhibitors geldanamycin and radicicol. Although this domain is essential for Hsp90 function, the molecular basis for adenosine nucleotide-dependent regulation of GRP94, the endoplasmic reticulum paralog of Hsp90, remains to be established. We report that bis-ANS (1,1'-bis(4-anilino-5-napthalenesulfonic acid), an environment sensitive fluorophore known to interact with nucleotide-binding domains, binds to the adenosine nucleotide-binding domain of GRP94 and thereby activates its molecular chaperone and peptide binding activities. bis-ANS was observed to elicit a tertiary conformational change in GRP94 similar to that occurring upon heat shock, which also activates GRP94 function. bis-ANS activation of GRP94 function was efficiently blocked by radicicol, an established inhibitory ligand for the adenosine nucleotide binding pocket. Confirmation of the N-terminal nucleotide binding pocket as the bis-ANS-binding site was obtained following covalent incorporation of bis-ANS into GRP94, trypsinolysis, and sequencing of bis-ANS-labeled limit digestion products. These data identify a ligand dependent regulation of GRP94 function and suggest a model whereby GRP94 function is regulated through a ligand-dependent conversion of GRP94 from an inactive to an active conformation.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *adenosine; *ADP; *ATP; *GRP94 --allosteric regulation; *GRP94 --peptide binding; *Hsp90; *N-ethyl-carboxamidoadenosine; *isothermal titration calorimetry --measurement method; *isothermal titration calorimetry --Analysis/Characterization Techniques: CB; *ATPase assay --activity assays; *ATPase assay --analytical method; * Fluoromax spectrofluorometer --equipment; * Fluoromax spectrofluorometer --Spex Industries; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --analytical method; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --electrophoretic techniques; *ligand interactions

2000

Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding.

Nicchitta, Christopher V.; Rosser, Meredith F. N.

Journal of Biological Chemistry, VOL. 275, NO. 30, July 28, 2000, PP. 22798-22805

X-ray crystallographic studies of the N-terminal domain of Hsp90 have identified an unconventional ATP binding fold, thereby inferring a role for ATP in the regulation of the Hsp90 activity. In this report, N-ethylcarboxamidoadenosine (NECA) was used to investigate the nucleotide binding properties of GRP94, the endoplasmic reticulum paralog of Hsp90. Whereas Hsp90 did not bind NECA, GRP94 bound NECA in a saturable manner with a Kd of 200 nM. NECA binding to GRP94 was efficiently blocked by geldanamycin and radicicol. Analysis of ligand binding stoichiometries by radioligand and calorimetric techniques indicated that GRP94 bound 1 mol of NECA/mol of GRP94 dimer. In contrast, GRP94 bound radicicol at a stoichiometry of 2 mol of radicicol/mol of GRP94 dimer. In (3H)NECA displacement assays, GRP94 displayed binding interactions with ATP, dATP, ADP, AMP, cAMP, and adenosine, but not GTP, CTP, or UTP. To accommodate the 0.5 mol of NE-CA:mol of GRP94 binding stoichiometry observed for the native GRP94 dimer, a model for allosteric regulation (negative cooperativity) of ligand binding is proposed. A hypothesis on the regulation of GRP94 conformation and activity by adenosine-based ligand(s) other than ATP and ADP is presented.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *adenosine; *ADP; *ATP; *GRP94 --allosteric regulation; *GRP94 --ligand binding; *Hsp90; *N-ethyl-carboxamidoadenosine; *isothermal titration calorimetry --measurement method; *isothermal titration calorimetry --Analysis/Characterization Techniques: CB; *ATPase assay --activity assays; *ATPase assay --analytical method; * Fluoromax spectrofluorometer --equipment; * Fluoromax spectrofluorometer --Spex Industries; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --analytical method; *SDS-PAGE SDS-polyacrylamide gel electrophoresis --electrophoretic techniques; *ligand interactions

2000

Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives.

Adams, Joseph A.; Ni, Da Qun; Shaffer, Jennife

Protein Science, VOL. 9, NO. 9, September, 2000 , PP. 1818-1827

The binding of the methylanthraniloyl derivatives of ATP (mant-ATP), ADP (mant-ADP), 2'deoxyATP (mant-2'deoxyATP), and 3'deoxyATP (mant-3'deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant-ADP is identical to that for ADP, while that for mant-ATP is approximately threefold higher than that for ATP. The dissociation constant for mant-3'deoxyATP is approximately fivefold higher than that for 3'deoxyATP while derivatization of 2'deoxyATP does not affect affinity. The time-dependent binding of mant-ATP, mant-2'deoxyATP, and mant-ADP, measured using stopped-flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant-ATP are equivalent substrates, as judged by reversed-phase chromatography, although the rate of phosphorylation is approximately 20-fold lower than the natural nucleotide. The kinetic data are consistent with a three-step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three-dimensional models for the enzyme.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *fluorescent adenosine derivatives --analysis; *fluorescent adenosine derivatives --enzyme substrate; *fluorescent adenosine derivatives --Sigma Chemicals; *protein kinase A --analysis; *protein kinase A --catalytic activity; *protein kinase A --molecular conformation; *protein kinase A --nucleotide binding; *protein kinase A --substrate specificity; *protein kinase A --transient state kinetics; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --Spectrum Analysis Techniques; *reversed-phase chromatography --purification method; *reversed-phase chromatography --Chromatographic Techniques; *stopped-flow fluorescence spectroscopy --analytical method; *stopped-flow fluorescence spectroscopy --Spectrum Analysis Techniques; *Applied PhotoPhysics stopped-flow spectrometer --equipment; *Applied PhotoPhysics stopped-flow spectrometer --Applied PhotoPhysics; * Fluoromax spectrofluorimeter --equipment

2000

Function of a conserved sequence motif in biotin holoenzyme synthetases.

Beckett, Dorothy; Kwon, Keehwan

Protein Science, VOL. 9, NO. 8, August, 2000, PP. 1530-1539

The biotin holoenzyme synthetases (BHS) are essential enzymes in all organisms that catalyze post-translational linkage of biotin to biotin-dependent carboxylases. The primary sequences of a large number of these enzymes are now available and homologies are found among all. The glycine-rich sequence, GRGRXG, constitutes one of the homologous regions in these enzymes and, based on its similarity to sequences found in a number of mononucleotide binding enzymes, has been proposed to function in ATP binding in the BHSs. In the Escherichia coli enzyme, the only member of the family for which a three-dimensional structure has been determined, the conserved sequence is found in a partially disordered surface loop. Mutations in the sequence have previously been isolated and characterized in vivo. In this work these single-site mutants, G115S, R118G, and R119W, of the E. coli BHS have been purified and biochemically characterized with respect to binding of small molecule substrates and the intermediate in the biotinylation reaction. Results of this characterization indicate that, rather than functioning in ATP binding, this glycine-rich sequence is required for binding the substrate biotin and the intermediate in the biotinylation reaction, biotinyl-5'-AMP. These results are of general significance for understanding structure-function relationships in biotin holoenzyme synthetases.

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques; *Escherichia coli (Enterobacteriaceae); *Bacteria; *Eubacteria; *Microorganisms; *biotin holoenzyme synthetase --analysis; *biotin holoenzyme synthetase --analyte; *biotin holoenzyme synthetase --conserved sequence motif; *biotin holoenzyme synthetase --flexible loop; *biotin holoenzyme synthetase --glycine motif; *biotin holoenzyme synthetase --ligand binding; *biotin holoenzyme synthetase --single-site mutants; *biotin holoenzyme synthetase --structure-activity relationships; *biotinyl-5'-AMP; *ATP --binding; *ATP --Sigma; *stopped-flow fluorescence --analytical method; *stopped-flow fluorescence --Spectrum Analysis Techniques; * Fluoromax-2 instrument --equipment; * Fluoromax-2 instrument --Instruments S.A. Inc.; *PC1 instrument --equipment; *PC1 instrument --ISS Inc.

2000

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): Induced tautomeric shifts on binding to enzyme, and enzymefwdarwligand fluorescence resonance energy transfer.

Kierdaszuk, Borys; Mondrak-Wojcik, Anna; Shugar, David.; Wierzchowski, Jacek

Biochimica et Biophysica Acta, VOL. 1476, NO. 1, Jan. 3,. 2000, PP. 109-128

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m1FA), N(2)-methylformycin A (m2FA) and N(6)-methylformycin A (m6FA), in the absence and presence of phosphate (Pi). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m6FA, independently of the presence of Pi. Fluorescence of m1FA in the complex increased only in the presence of Pi, while the weak fluorescence of FB appeared unaffected, independently of Pi. Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m6FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m2FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme-FA, or enzyme-m6FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of Pi. Complex formation with m6FA in the absence of Pi led to a shift of the amino-imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of Pi, the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant Kd = 0.5 +- 0.1 muM, two orders of magnitude lower than that for m6FA in the absence of Pi (Kd = 46 +- 5 muM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values (fa) varied from 0.31 for m1FA to 0.70 for FA, with negative cooperative binding of m1FA and FB, and non-cooperative binding of FA and m6FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes (ltbbractaurtbbrac) affected by their interactions. Complex formation resulted in a 2-fold increase in ltbbractaurtbbrac of FA, and a 2-fold decrease in ltbbractaurtbbrac of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies..

DESCRIPTOR(S)- *Enzymology (Biochemistry and Molecular Biophysics); *Methods and Techniques.; *Escherichia coli (Enterobacteriaceae).; *Bacteria; *Eubacteria; *Microorganisms.; *formycin A --analysis; *formycin A --enzyme binding; *formycin A --enzyme inhibitor; *formycin A --tautomeric shifts; *formycin A --N-methyl analogues; *formycin A --Sigma Chemical Co.; *formycin B --analysis; *formycin B --enzyme binding; *formycin B --enzyme inhibitor; *formycin B --tautomeric shifts; *formycin B --N-methyl analogues; *formycin B --Sigma Chemical Co.; *purine nucleoside phosphorylase EC 2.4.2.1 --analysis; *purine nucleoside phosphorylase EC 2.4.2.1 --complex formation; *purine nucleoside phosphorylase EC 2.4.2.1 --fluorescence quenching; *purine nucleoside phosphorylase EC 2.4.2.1 --inhibition.; *N(1)-methylformycin A --analysis; *N(1)-methylformycin A --enzyme binding; *N(1)-methylformycin A --enzyme inhibitor; *N(1)-methylformycin A --tautomeric shifts; *N(1)-methylformycin A --N-methyl analogue; *N(2)-methylformycin A --analysis; *N(2)-methylformycin A --enzyme binding; *N(2)-methylformycin A --enzyme inhibitor; *N(2)-methylformycin A --tautomeric shifts; *N(2)-methylformycin A --N-methyl analogue; *N(6)-methylformycin A --analysis; *N(6)-methylformycin A --enzyme binding; *N(6)-methylformycin A --enzyme inhibitor; *N(6)-methylformycin A --tautomeric shifts; *N(6)-methylformycin A --N-methyl analogue; *steady-state emission spectroscopy --analytical method; *steady-state emission spectroscopy --spectroscopic techniques: CB; *time-resolved emission spectroscopy --analytical method; *time-resolved emission spectroscopy --spectroscopic techniques: CB.; *IBH time-resolved spectrofluorimeter --equipment; *IBH time-resolved spectrofluorimeter --IBH Consultants; *Spex FluoroMax spectrofluorimeter --equipment; *enzyme-ligand interaction.

2000

DNA damage by thiol-activated neocarzinostatin chromophore at bulged sites.

Goldberg, Irving H.; Gu, Feng; Xi, Zhen

Biochemistry, VOL. 39, NO. 16, April 25, 2000, PP. 4881-4891

Bulge structures in nucleic acids are of general biological significance and are potential targets for therapeutic drugs. It has been shown in a previous study that thiol-activated neocarzinostatin chromophore is able to cleave duplex DNA selectively at a position opposite a single unpaired cytosine or thymine base on the 3' side. In this work, we studied in greater detail the nature of this type of cleavage and the basis for the selectivity of the bulge site cleavage over the usual strand cleavage at a T site in the duplex region by using duplexes containing an internal control and a bulge, which is composed of different types and number of bases. Experimental results indicated that the bulge-induced cleavage is initiated by 5' hydrogen abstraction and is greatly affected by the base composition of the bulge. A single-base bulge, especially when containing a purine, yields higher efficiency and greater selectivity for the bulge-induced cleavage. In particular, a single adenine base gives rise to the highest cleavage yield and provides over 20 times greater selectivity for cleavage at the bulge site compared with the internal control site in duplexes. The binding dissociation constants of postactivated drug for a stem-loop structure containing a one- or two-base bulge in the stem, measured by fluorescence quenching, show that the binding is about 3-4 times stronger for bulge-containing duplexes than for perfect hairpin duplexes. For RNAcntdotDNA hybrid duplexes, where the DNA is the target strand and the RNA is the bulge-containing strand, bulge-induced cleavage was observed, although at low yield. On the other hand, when RNA is the nonbulge strand, no bulge-induced cleavage was found. When the reaction is performed in the absence of oxygen, the major product is a covalent adduct, and it is at the same location as the cleavage site under aerobic conditions.

DESCRIPTOR(S)- *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Pharmacology; *neocarzinostatin chromophore --analysis; *neocarzinostatin chromophore --antitumor antibiotic; *neocarzinostatin chromophore --pharmaceutical; *neocarzinostatin chromophore --thiol-activated; *DNA --analysis; *DNA --bulge-induced cleavage; *DNA --damage; *fluorescence quenching --analytical method; *fluorescence quenching --Spectrum Analysis Techniques; *SPEX Fluoro Max-2 --equipment

2000

Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta.

Kuhlmann, Juergen; Goerlich, Dirk; Nowak, Christine; Villa Braslavsky, Carolina I.; Wittinghofer, Alfred

Biochemistry, VOL. 39, NO. 38, September 26, 2000, PP. 11629-11639

The cytoplasmic disassembly of RancntdotGTPcntdotimportin and RancntdotGTPcntdotexportincntdotcargo complexes is an essential step in the corresponding nuclear import and export cycles. It has previously been shown that such disassembly can be mediated by RanBP1 in the presence of RanGAP. The nuclear pore complex protein RanBP2 (Nup358) contains four Ran-binding domains (RanBDi) that might function like RanBP1. We used biophysical assays based on fluorescence-labeled probes and on surface plasmon resonance to investigate the dynamic interplay of Ran in its GDP- and GTP-complexed states with RanBDis and with importin-beta. We show that RanBP1 and the four RanBDis from RanBP2 have comparable affinities for RancntdotGTP (108-109 M-1). Deletion of Ran's C-terminal 211DEDDDL216 sequence weakens the interaction of RancntdotGTP with RanBPis approximately 2000-fold, but accelerates the association of RancntdotGTP with importin-beta 10-fold. Importin-beta binds RancntdotGTP with a moderate rate, but attains a high affinity for Ran (KD = 140 pM) via an extremely low dissociation rate of 10-5 s-1. Association with Ran is accelerated 3-fold in the presence of RanBP1, which presumably prevents steric hindrance caused by the Ran C-terminus. In addition, we show that the RanBDis of RanBP2 are full equivalents of RanBP1 in that they also costimulate RanGAP-catalyzed GTP hydrolysis in Ran and relieve the GTPase block in a RancntdotGTPcntdottransportin complex. Our data suggest that the C-terminus of Ran functions like a loose tether in RancntdotGTP complexes of importins or exportins that exit the nucleus. This flag is then recognized by the multiple RanBDis at or near the nuclear pore complex, allowing efficient disassembly of these RancntdotGTP complexes.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Cell Biology; *Methods and Techniques; *importin-beta --analysis; *importin-beta --Ran-binding domain; *Ran --analysis; *Ran --carboxyl-terminus; *Ran --molecular structure; *Ran --nuclear pore complex; *RanBP2 --analysis; *RanBP2 --Ran-binding domain; *biophysical assay --bioassay method; *biophysical assay --Bioassays/Physiological Analysis; *surface plasmon resonance analysis --analytical method; *surface plasmon resonance analysis --Spectrum Analysis Techniques; *SPEX Fluoromax fluorescence spectrometer --equipment

2000

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: Spectroscopic and calorimetric studies.

Makhatadze, George I.; Jacquot, Jean-Pierre; Lemaire, Stephane D.; Richardson, John M., III

Biochemistry, VOL. 39, NO. 36, September 12, 2000, PP. 11154-11162

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change (6.0 +- 1.0 kJ/(molcntdotK)), suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degreeC, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *Chlamydomonas reinhardtii (Chlorophyta); *Algae; *Microorganisms; *Nonvascular Plants; *Plants; *thioredoxin h --analysis; *differential scanning calorimetry --analytical method; *differential scanning calorimetry --Bioassays/Physiological Analysis; *far-UV circular dichroism --analytical method; *far-UV circular dichroism --circular dichroism; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --fluorometry: CB; *near-UV circular dichroism --analytical method; *near-UV circular dichroism --circular dichroism; * FluoroMax spectrofluorometer --equipment; *Jasco J-20 spectropolarimeter --equipment; *Jasco J-20 spectropolarimeter --Jasco; *VP-differential scanning calorimeter --equipment; *VP-differential scanning calorimeter --Microcal

2000

Detection of phosphopeptides by fluorescence polarization in the presence of cationic polyamino-acids: application to kinase assays.

Coffin, J.; Latev, M.; Bi, X. H.; Nikiforov, T. T.

Analytical Biochemistry, VOL. 278, NO. 2, 2000-02-15, PP. 206-212

The discrimination of fluorescently labelled phosphorylated peptides from their nonphosphorylated analogues using fluorescence polarization in the presence of cationic polyamino-acids such as polylysine and polyarginine was evaluated. Fluorescence polarization measurements were performed on a FluoroMax-2 instrument (Instruments, S. A. ) with excitation and emission at 490 nm and 520 nm, respectively, for fluorescein. Five different fluorescein-labelled peptides, containing substrate recognition sites for three different serine/threonine kinases (protein kinase A, protein kinase B small alpha , and protein kinase C) were tested. Preferential binding of the phosphorylated peptides to the cationic polyamino-acids was detected by an increase in the fluorescence polarization signal. This approach provides an alternative method for kinase assays. The method was applied to K K M determinations of kinase enzymes for their substrates and to K 1 determinations for a protein kinase A inhibitor (H-89).

2000

Conformation of the isolated Cepsilon3 domain of IgE and its complex with the high-affinity receptor, FcepsilonRI.

Gould, Hannah J.; Ghirlando, Rodolfo; Henry, Alistair J.; McDonnell, James M.; Sutton, Brian J.

Biochemistry, VOL. 39, NO. 25, June 27, 2000, PP. 7406-7413

Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, FcepsilonRI, on the surface of mast cells and basophils. Previous work has implicated the third domain of the constant region of the epsilon-heavy chain (Cepsilon3) in binding to FcepsilonRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the Cepsilon3 and Cepsilon4 domains. We have expressed the isolated Cepsilon3 in Escherichia coli, measured its affinity for FcepsilonRI, and examined its conformation alone and in the complex with FcepsilonRI. Sedimentation equilibrium in the analytical centrifuge reveals that this products is a monomer. The kinetics of binding to an immobilized fragment of the FcepsilonRI alpha-chain, measured by surface plasmon resonance, yields an affinity constant Ka = 5 X 106 M-1, as compared with 4 X 109 M-1 for IgE. The circular dichroism spectrum and measurements of fluorescence as a function of the concentration of a denaturant do not reveal any recognizable secondary structure or hydrophobic core. On binding to the FcepsilonRI alpha-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of Cepsilon3 is unchanged in the complex. Thus the isolated Cepsilon3 domain is sufficient for binding to FcepsilonRI, but with lower affinity than IgE. This may be due to the loss of its native immunoglobulin domain structure or to the requirement for two Cepsilon3 domains to constitute the complete binding site for FcepsilonRI or to a combination of these factors.

DESCRIPTOR(S)- *Immune System (Chemical Coordination and Homeostasis); *Methods and Techniques; *Molecular Genetics (Biochemistry and Molecular Biophysics); *Escherichia coli (Enterobacteriaceae) --expression vector; *Bacteria; *Eubacteria; *Microorganisms; *immunoglobulin E --analysis; *immunoglobulin E --expression; *immunoglobulin E --receptor complexation; *immunoglobulin E --third domain; *analytical centrifugation --analytical method; *analytical centrifugation --Molecular Biology Techniques & Chemical Characterization; *circular dichroism spectroscopy --analytical method; *circular dichroism spectroscopy --Spectrum Analysis Techniques; *fluorescence emission spectroscopy --analytical method; *fluorescence emission spectroscopy --Spectrum Analysis Techniques; *Beckman Optima XL-A analytical centrifuge --equipment; *Beckman Optima XL-A analytical centrifuge --Beckman; *ELISA --detection method; *ELISA --detection/labeling techniques; * Fluoromax fluorimeter --equipment; *Jobin-Yvon CD6 spectrophotometer --equipment

2000

Charged membrane surfaces impede the protein-mediated transfer of glycosphingolipids between phospholipid bilayers.

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

Biochemistry, VOL. 39, NO. 5, Feb. 8,. 2000, PP. 1067-1075

A lipid transfer protein that facilitates the transfer of glycolipids between donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa, pI 9.0) catalyzes the high specificity transfer of lipids that have sugars beta-linked to either a ceramide or a diacylglycerol backbone, such as simple glycolipids and gangliosides, but not the transfer of phospholipids, cholesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled galactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of negatively charged lipid at 5 or 10 mol % into the donor vesicles significantly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was negative charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (pI apprxeq 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques.; *galactosylceramide; *gangliosides; *glycolipid transfer protein; *glycosphingolipids; *phospholipid bilayers.; *fluorescence resonance energy transfer assay; *kinetic analysis; *protein purification; *thin-layer chromatography.; *DEAE Sephacel minicolumn; *SPEX Fluoromax instrument; *charged membrane surfaces; *pH.

2000

Assessing adrenergic receptor conformation using chemically reactive fluorescent probes.

Dam Jensen, Anne; Gether, Ulrik

Methods in Molecular Biology, VOL. 126, 2000, PP. 345-361

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques; *baculovirus (Baculoviridae) --gene vector; *Sf9 cell line (Lepidoptera); *Animal Viruses; *Animals; *Arthropods; *Insects; *Invertebrates; *Microorganisms; *Viruses; *adrenergic receptors; *fluorescent probes; *rhodopsin; *tryptophan; *G-protein coupled receptor; *G-protein-alpha-subunit; *alprenolol affinity chromatography --liquid chromatography; *alprenolol affinity chromatography --protocol; *alprenolol affinity chromatography --purification method; *cell culture --cell culture method; *cell culture --protocol; *cell culture --Cell Culture Techniques; *fluorescence spectroscopy --analytical method; *fluorescence spectroscopy --protocol; *fluorescence spectroscopy --spectroscopic techniques: CB; *immunoaffinity chromatography --affinity chromatography; *immunoaffinity chromatography --protocol; *immunoaffinity chromatography --purification method; *immunoaffinity chromatography --Recombinant Protein Protocols; *nickel affinity chromatography --liquid chromatography; *nickel affinity chromatography --protocol; *nickel affinity chromatography --purification method; *protein purification --protocol; *protein purification --purification method; *protein purification --Isolation/Purification Techniques: CB; *radioligand binding assay --binding assays; *radioligand binding assay --detection method; *radioligand binding assay --detection/labeling techniques; *radioligand binding assay --protocol; *EPR spectroscopy --analytical method; *EPR spectroscopy --protocol; *EPR spectroscopy --spectroscopic techniques: CB; *Poly prep columns --laboratory equipment; *Poly prep columns --Bio-Rad; *Sephadex G50 resin --laboratory equipment; *Sephadex G50 resin --Pharmacia; *SPEX Fluoromax-2 spectrofluorometer --laboratory equipment; *SPEX Fluoromax-2 spectrofluorometer --SPEX; *protein conformation; *protein structure; *Book Chapter

2000

Acute ethanol modulates glutamate induced (Ca2+)i transients and increase cell volume in astroglial primary cultures from rat cerebral cortex.

Allansson, L.; Hansson, E.

Alcoholism Clinical and Experimental Research, VOL. 24, NO. 5 Supplement, May, 2000, PP. 221A.

DESCRIPTOR(S)- *Nervous System (Neural Coordination); *Toxicology; *rat (Muridae); *Animals; *Chordates; *Mammals; *Nonhuman Mammals; *Nonhuman Vertebrates; *Rodents; *Vertebrates; *astrocyte --nervous system; *astroglia --cell volume; *astroglia --nervous system; *cerebral cortex --nervous system; *calcium --transients; *ethanol --toxin; *fura-2/AM; *glutamate; *Fluo-3/AM; *SPEX Fluoromax imaging --imaging method; *Meeting Abstract

2000

A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes.

Pispisa, Basilio; Marchiori, Fernando; Palleschi, Antonio; Polese, Alessandra; Stella, Lorenzo; Toniolo, Claudio.; Venanzi, Mariano

Biopolymers, VOL. 53, NO. 2, Feb.,. 2000, PP. 169-181

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)n-Trp-(Aib)r-OtBu (T(Aib)nTrp), where n + r = 4, and Toac is a nitroxide spin-labeled Calpha,alpha-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-(Orn-((Aib)2-Trp-(Aib)2-Z)-Asp-((Aib)2-Toac-(Aib)2-OtBu)) (T-cyclo-Trp), was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 310-helical conformation, as earlier determined by x-ray diffraction for T(Aib)3Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution..

DESCRIPTOR(S)- *Biochemistry and Molecular Biophysics; *Methods and Techniques.; *amino acids --analysis; *linear peptides --analysis; *linear peptides --molecular properties; *nitroxide derivative --analysis; *nitroxide derivative --molecular probe; *peptide templates --analysis; *peptide templates --molecular properties; *peptides --analysis; *peptides --molecular properties; *tryptophan --analysis; *tryptophan --molecular probe.; *circular dichroism --analytical method; *circular dichroism --spectroscopic techniques: CB; *steady-state fluorescence spectra --analytical method; *steady-state fluorescence spectra --spectroscopic techniques: CB.; *IR spectrophotometry --analytical method; *IR spectrophotometry --spectrophotometry: CB; *SPEX Fluoromax spectrofluorometer --equipment; *SPEX Fluoromax spectrofluorometer --SPEX; *dipole-dipole interaction mechanisms --analysis; *molecular mechanics calculations; *molecular modeling; *solution chemistry --applications.