RESUMO
The excitation-emission spectra of flavin mononucleotide (FMN) were measured in rigid PVA films for concentrations ranging from 6.92 x 10(-4)M to 1.03 M. The theoretical three-linear decomposition of the excitation-emission spectra indicated the presence of two absorption and emission centers corresponding to FMN monomer and dimer, respectively. The component of the fluorescence profile corresponding to the FMN monomer has a large negative part which is the mirror image of the emission band profile of the dimer. The elimination of this part by taking a linear combination of the emission components of the monomer and of the dimer resulted in emission spectrum, which is in a very good agreement with the monomer spectrum measured directly. The appearance of a negative part of the monomer emission profile obtained by trilinear decomposition of the emission-absorption spectra of FMN can be explained in terms of the non-radiative reverse energy transfer from the FMN dimers to the FMN monomers. The presented results confirm that the FMN molecules in rigid PVA form dimers but not higher order aggregates. Moreover, they enable to obtain fluorescence spectra of dimers and suggest that FMN dimers may take part in the process of non-radiative energy transfer occurring in photoreception phenomena.
Assuntos
Mononucleotídeo de Flavina/química , Dimerização , Transferência de Energia , Álcool de Polivinil , Espectrometria de FluorescênciaRESUMO
We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distances was found for free melittin, which is consistent with that expected for the random coil state, characterized by a Gaussian width (full width at half maxima) of 28.2 A. In contrast, narrow distance distributions were found for melittin complexed with CaM, 8.2 A, or with POPC vesicles, 4.9 A. A somewhat wider distribution was found for the melittin complex with TnC, 12.8 A, suggesting the presence of heterogeneity in the mode of binding between melittin and TnC. For all the complexes the mean Trp-19 to dansyl distance was near 20 A. This value is somewhat smaller than expected for the free alpha-helical state of melittin, suggesting that binding with CaM or TnC results in a modest decrease in the length of the melittin molecule.
Assuntos
Calmodulina/metabolismo , Meliteno/química , Meliteno/metabolismo , Fosfolipídeos/metabolismo , Troponina/metabolismo , Triptofano/química , Fenômenos Químicos , Físico-Química , Compostos de Dansil/química , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Espectrofotometria , Troponina CRESUMO
Papain activity was studied in water-organic solvent mixtures using the fluorogenic substrate Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. The increase of organic solvent (MeOH, EtOH, iPrOH, TFE, MeCN, (MeO)2Et and DMF) concentration in the mixture caused a substantial decrease the initial rate of papain-catalyzed hydrolysis. Moreover, the number of papain active sites decreased with the increase of DMF and MeOH concentration.
Assuntos
Papaína/química , Sequência de Aminoácidos , Sítios de Ligação , Dimetilformamida/química , Relação Dose-Resposta a Droga , Hidrólise , Cinética , Dados de Sequência Molecular , Biossíntese Peptídica , Espectrometria de FluorescênciaRESUMO
Papain activity in a buffer containing Me2SO was studied using fluorogenic substrates. It was found that the number of active sites of papain decreases with increasing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain incubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an increase of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michaelis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of k(cat)/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis k(cat).
Assuntos
Dimetil Sulfóxido/farmacologia , Papaína/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/química , Hidrólise , Cinética , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/química , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Fluorescence resonance energy transfer (FRET) is a technique widely used in studies of interchromophoric distances in biomolecules such as peptides, proteins and nucleic acids. FRET is especially useful in determination of conformational changes caused by a solvent, presence of denaturing agents, diffusion and other external factors. Precision of interchromophoric distances obtained using the FRET technique is comparable with that of low-resolution X-ray diffraction and NMR data. Comparison of FRET results with the crystal structure for several proteins is reviewed. Moreover, the effect of the orientation factor kappa2 value on FRET results and determinants of kappa2 are discussed.
Assuntos
Conformação Proteica , Espectrometria de FluorescênciaRESUMO
The lifetime distribution calculations were applied to study the influence of configuration of amino-acid residues in positions 2 and 3 on changes in conformation of the peptide chain of cyclic analogues of enkephalins containing a fluorescence energy donor and acceptor in different solvents. In all the solvents studied the lifetime distributions were bimodal. This testified to the presence of two families of conformations. In this paper the relationship between the population of each conformation and configuration of the residues in position 2 and 3, and the solvent used is discussed.
Assuntos
Encefalinas/química , Sequência de Aminoácidos , Transferência de Energia , Peptídeos Cíclicos/química , Conformação Proteica , Solventes , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Lifetime distribution analysis were performed to study the influence of Leu configuration in position 5 on changes of the peptide chain of cyclic analogues of enkephalins containing a fluorescence donor and acceptor in different solvents. The configuration change of Leu5 in all the analogues of enkephalins studied which contain donor-acceptor pairs has no apparent influence on Trp lifetime distributions. In contrast, there is a significant solvent effect on the shape of lifetime distribution.
Assuntos
Encefalinas/química , Leucina/química , Modelos Químicos , Modelos Estatísticos , Conformação Proteica , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.
Assuntos
Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/química , Animais , Domínio Catalítico , Sequência Conservada , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
We used frequency-domain fluorometry to measure intensity and anisotropy decay of indole fluorescence in cyclohexane/ethanol mixtures at 20 degrees C. In 100% cyclohexane or 100% ethanol the intensity decay of indole appears to be a single exponential with decay times of 7.66 and 4.10 ns, respectively. In cyclohexane containing a small percentage of ethanol (up to 10%), we observed increased heterogeneity in intensity decay, resulting in a 10-fold increase in chi 2R for the single-exponential fit, as compared with the double-exponential model. We obtained comparable or better fits using unimodal Lorentzian and Gaussian lifetime distributions (two floating parameters) than for the two-exponential model (three floating parameters). We believe that the distribution of decay times reflects a range of indole solvation states in the dominately nonpolar solutions. This result suggests that a variety of hydrogen-bonding configurations could be one origin of the distributions of decay times observed for tryptophan emission from proteins. We also measured rotational diffusion of indole in cyclohexane, ethanol and its mixtures at 20 degrees C. The picosecond correlation times required that the mean decay times be decreased by acrylamide quenching (in ethanol) or energy transfer (in cyclohexane). In ethanol we observed nearly isotropic rotation of indole; in cyclohexane we obtained two correlation times of 17 and 73 ps. The shorter correlation time in cyclohexane appears to be due to the slip boundary condition, which was found to be progressively eliminated by small percentages of ethanol. Hence, hydrogen-bonding interactions appear to have a substantial effect on the rotational dynamics of indole.
Assuntos
Cicloexanos , Etanol , Indóis , Espectrometria de Fluorescência/métodos , Matemática , Modelos TeóricosRESUMO
The technique of 10 GHz frequency-domain fluorometry was used to resolve the complex picosecond intensity and anisotropy decays of the tyrosyl emission of [Leu5] enkephalin. Enhanced resolution of anisotropy decay was obtained by using acrylamide quenching of the tyrosyl fluorescence and global analysis of the frequency-domain anisotropy data obtained with different amounts of acrylamide. The data indicates a 44 ps correlation time for local tyrosine motions, and a 219 ps correlation time for overall rotational diffusion of the pentapeptide. Our data are consistent with an initial loss of fluorescence anisotropy from r0 = 0.4 to a value of r0 = 0.326 occurring during the first two picoseconds after excitation.
Assuntos
Encefalina Leucina/química , Tirosina/química , Acrilamidas/química , Sequência de Aminoácidos , Cinética , Dados de Sequência Molecular , Espectrometria de Fluorescência , Temperatura , TermodinâmicaRESUMO
Frequency-domain fluorescence spectroscopy was employed to examine the decays of tryptophan in Boc-Trp-Met-Asp-Phe-NH2 (donor) and (Formula: see text) (donor-acceptor pair). The efficiency of energy transfer in the thiopeptide amounted to 60%. The measured dispersion of fluorescence decay times was used to recover the donor-acceptor distance distribution. The parameters of the Gaussian distance distribution obtained for this peptide (r, the mean distance (9 A); hw, the halfwidth (25 A)) indicate the lack of a distinct favorable conformation.
Assuntos
Pentagastrina/análogos & derivados , Compostos de Sulfidrila , Transferência de Energia , Pentagastrina/síntese química , Conformação Proteica , Espectrometria de Fluorescência/métodos , Compostos de Sulfidrila/síntese química , TriptofanoRESUMO
We investigated the influence of end-to-end diffusion on intramolecular energy transfer between a naphthalene donor and dansyl acceptor linked by polymethylene chain. A range of viscosities from 0.6 to 200 cP were obtained using propylene glycol at different temperatures (0-80 degrees C) and methanol at 20 degrees C. The intensity decays of naphthalene were measured in the frequency domain. Several theoretical models, including distance distributions, were used to fit the data. The results indicate that end-to-end diffusion of flexible donor-acceptor pairs can be detected and quantified using frequency-domain fluorometry, even in the presence of a distribution of donor-to-acceptor distances.
Assuntos
Transferência de Energia , Difusão , Cinética , Matemática , Metanol/química , Propilenoglicol , Propilenoglicóis/química , Espectrometria de FluorescênciaRESUMO
We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.
Assuntos
Meliteno/química , Metanol , Solventes , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.
Assuntos
Nuclease do Micrococo/química , Triptofano/química , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Temperatura Baixa , Conformação Proteica , Solventes/química , Espectrometria de Fluorescência/métodos , Staphylococcus aureus/enzimologia , Temperatura , Termodinâmica , Fatores de TempoRESUMO
We measured distance distributions and end-to-end diffusion coefficients of donor-acceptor pairs linked by a flexible methylene chain using frequency-domain fluorescence energy transfer measurements. The donor was an indole group, and two acceptors with different Förster distances were used. The uncertainties in the recovered parameters describing the end-to-end distance distribution and diffusion coefficient were rather large when each donor-acceptor pair was analyzed separately. It was not possible to resolve distance distributions in the presence of intra-molecular diffusion when the Förster distance was comparable to the mean and half-width of the distribution. Global analysis using two acceptors dramatically improved the resolution. Surprisingly, the Förster distances need not be very different, and a 20% difference between the two R0 values resulted in substantial improvements in resolution. Both the simulations and the experiments suggest the benefit of using global analysis with different Förster distances to obtain reliable distance distribution parameters in the presence of diffusion.
Assuntos
Benzenossulfonatos/química , Modelos Biológicos , Ácidos Mirísticos/química , Nitrobenzenos/química , Triptaminas/química , Benzenossulfonatos/metabolismo , Difusão , Transferência de Energia , Polarização de Fluorescência , Substâncias Macromoleculares , Conformação Molecular , Estrutura Molecular , Ácidos Mirísticos/metabolismo , Nitrobenzenos/metabolismo , Espectrometria de Fluorescência , Triptaminas/metabolismoRESUMO
We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.
Assuntos
Modelos Teóricos , Conformação Molecular , Matemática , Probabilidade , Conformação Proteica , Espectrometria de Fluorescência/métodosRESUMO
The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.
Assuntos
Cisteína/química , Subfragmentos de Miosina/química , Ligantes , Conformação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Galanin (GAL) is a 29 amino acid C-terminally aminated linear neuropeptide showing diverse biological activities. The N-terminal (1-15)GAL-NH2 fragment was shown to have a very high affinity to the galanin receptor. In this work we describe the results of a combined fluorescence and Monte Carlo studies, the latter carried out using the ECEPP/3 force field with and without including hydration, on the (1-15)GAL-NH2 fragment. Using the time-domain technique we measured fluorescence decay times of the tyrosine residue in position 9. Based on the Forster energy transfer theory we calculated the distance and distance distribution between the Trp2 (acceptor) and Tyr9 (donor) aromatic side chains. The distance obtained was about 10.5 angstrom and half-width, hw, of the distance distribution was 5.6 angstrom. This results were found to be in good agreement with the chromophore distances calculated for the low-energy solution confirmations obtained in Monte Carlo simulations. All the low-energy conformations obtained in the absence of water were almost all-helical with the exception of a few C-terminal residues. In contrast, none of the low-energy solution conformations contained any significant amount of secondary structure. These findings are in agreement with the results of earlier CD and NMR conformational studies of galanin in water and non-aqueous solvents. On the other hand, the conformations obtained in the presence of water turned out to be largely compact in the N-terminal hydrophobic part. This explains the relatively short distance between chromophores and narrow distance distribution obtained in fluorescence measurements.
Assuntos
Galanina/química , Fragmentos de Peptídeos/química , Amidas/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Método de Monte Carlo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de FluorescênciaRESUMO
We describe a novel application of frequency-domain fluorometry which allows resolution of the decay times and emission spectra of samples which display multi-exponential decay kinetics. This method does not require any previous knowledge about the decay times or any assumptions about the shape of the emission spectra. We record the wavelength-dependent phase angles and modulations (phase angle and modulation spectra) using a number of light modulation frequencies. The data is analyzed by non-linear least-squares to recover the emission spectra and their associated decay times. Phase and modulation spectra (PM Spec) were used to recover the emission spectra associated with the two decay times of tryptophan at pH = 7 (0.54 and 3.44 ns). The emission spectra of these components are centered at 340 and 355 nm, respectively, with the amplitude of the 0.54 ns component contributing 6% to the total emission. These results are in agreement with previous time-resolved studies by Szabo and Rayner [J. Am. Chem. Soc. 102, 554-563 (1980)]. Control experiments were performed on mixtures of N-acetyl-L-tryptophanamide (NATA) and PPD, which demonstrate our ability to recover the spectra and decay times from two component mixtures. NATA itself displayed a single decay time and only one emission spectrum.
Assuntos
Espectrometria de Fluorescência/métodos , Triptofano/análise , Oxazóis/análise , Fotoquímica , Triptofano/análogos & derivadosRESUMO
We report measurements of the site-to-site diffusion coefficients in proteins and model compounds, which were measured using time-dependent energy transfer and frequency-domain fluorometry. The possibility of measuring these diffusion coefficients were shown from simulations, which demonstrate that donor (D)-to-acceptor (A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of donor-to-acceptor distances. For decay times typical of tryptophan fluorescence, the simulations indicate that D-A diffusion coefficients can be measured ranging from 10(-7) to 10(-5) cm2/s. This possibility was verified by studies of a methylene-chain linked D-A pair in solutions of varying viscosity. The D-A diffusion was also measured for two labeled peptides and two proteins, melittin and troponin I. In most cases we used global analysis of data sets obtained with varying amounts of collisional quenchers to vary the donor decay time. Unfolding of troponin I results in more rapid D-A diffusion, whereas for melittin more rapid diffusion was observed in the alpha-helical state but over a limited range of distances.