首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 140 毫秒
1.
We present results of time resolved fluorescence measurements performed in Tryptophan (Trp) derivatives and Trp-containing peptides in the pH range 3.0-11.0. For each compound a set of decay profiles measured in a given range of pH values was examined as a whole, using the global analysis technique. The data were fitted to two or three lifetime components and the analysis allowed the monitoring of the changes in the concentration of the different species contributing to the total fluorescence in that pH interval. The decay components were sensitive to the ionization state of groups neighboring the indol ring, and pK values for the equilibrium between protonated and deprotonated species were obtained from the preexponential factor of the lifetime components. In Trp, protonation of the amino terminal of the rotamer having electron transfer rate comparable to fluorescence decay rates was responsible for the interconvertion of a long lifetime component, to the 2.9 ns component usually observed in neutral pH. Trpbond;X peptides also have a single rotamer dominating the decay that is quenched by NH(3) (+). X-Trp peptides seem to be conformationally less restricted, and it is possible that rotamers interconvertion occur in high pH, increasing the population of nonquenched rotamers. Interconvertion between rotameric conformations of Trp are also present in the titration of ionizable groups in the side chain of peptides like His-Trp and Glu-Trp and control of pH is essential to the correct interpretation of fluorescence data in the study of peptides having such groups near to the Trp residue.  相似文献   

2.
The fluorescence quantum yield and the fluorescence decay of aqueous solutions of derivatives containina a single tyrosine residue have been measured at different pH. In these derivatives tyrosine was substituted on its amino end (series I) or/and, on its carboxyl end (series II), by acyl, amino or amino acyl groups. The fluorescence decays of series I derivatives are monoexponential regardless to the ionization state of their amino group. Upon deprotonation of the α-amino group, the quantum yields and the lifetimes increase in the case of dipeptides, and slightly decrease, for the tripeptides. The quantum yield and the lifetime increase with the side chain length of the aliphatic residue adjacent to the tyrosine residue, (the fluorescence of Val Tyr anion being identical to that of free Tyrosine). Quite different is the behavior of series II derivatives: their decays at pH 5.5 must be described by two exponential terms, one of them decaying with a short time constant (about 0.5 ns) and little side chain effect is observed. The fluorescence intensity increases upon deprolonalion of the α-amino proup (though to a lesser extent than for series I derivatives); a nearly monoexponential decay is observed at basic pH for dipeptides. but not for tyrosine amide, amide or dipeptides, or tripeptides. The following interpretation of our results is proposed: fluorescence quenching occurs in molecular conformations in which a peptide carbonyl can come in contact with the phenolic chromophore. This condition depends mainly on the value of the angle x1 which determines the conformation of the tyrosyl residue around its Cα-Cβ bond. It appears that the rotamer in which quenching occurs are not the same for series I and series II derivatives, which can explain the different behavior of these two kinds of compounds. The interpretation of the fluorescence properties is developed taking into account on one side the relative population of the rotamers in the ground state, which is given by studies of crystals and of solutions, and on the other side the possibility of an exchange between these rotamers during the excited state time. In this scheme the protonated α-amino groups would act to reinforce the quenching efficiency of the carbonyl. At last it is found that the radiative lifetime of the phenolic chromophore is the same for all the compounds studies.  相似文献   

3.
The dynamic and static properties of the fluorescence of a pyrene-introduced oligonucleotide 16 mer and its hybrid with a target 32 mer. Their fluorescence quantum yields (< 1%) were much weaker than that of unsubstituted pyrene and their fluorescence lifetime of the major decay components were less than 1 ns. The rapid fluorescence quenching was due to the interaction between the fluorophore and bases in the oligonucleotides. The fluorescence of pyrene was quenched efficiently by TMP and slightly by AMP. The quenching by CMP and GMP were the intermediate case.  相似文献   

4.
The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.  相似文献   

5.
We have inserted a tryptophan (F77W) in the core of the regulatory domain of cardiac troponin C (cNTnC), and previously determined the structure of this mutant with and without the cosolvent trifluoroethanol (TFE). Interestingly, the orientations of the indole side chain of the Trp are in opposite directions in the two structures (Julien et al., Protein Sci 2009; 18:1165-1174). Fluorescence decay experiments for single Trp-containing proteins often show several lifetimes, which have been interpreted as reflecting conformational heterogeneity of the Trp side chain resulting from different rotamers. To test this interpretation, we monitored the effect of TFE on wild type, F77W and F77W-V82A calcium-saturated cNTnC using 2D (13)C-HSQC NMR and time-correlated single photon counting fluorescence spectroscopies. The time dependence of the Trp fluorescence decay was fit with three lifetimes. Addition of TFE caused a gradual, but limited decrease of the lifetimes due to dynamic quenching. For F77W cNTnC, the amplitude fractions of the lifetimes also changed upon addition of TFE-the long lifetime increased from 13 to 29%, while the middle lifetime decreased from 63 to 50% and the short lifetime remained relatively unchanged. For F77W-V82A cNTnC, comparable NMR changes are observed, confirming the switch in rotamer conformation, but only much smaller changes in fluorescence decay parameters were detected. These data indicate that the balance between the rotamer states can be changed without changing the lifetime amplitude fractions appreciably, and suggest that the environment(s) of the indole ring, responsible for the different lifetimes, can result from factors other than the intrinsic rotamer state of the tryptophan.  相似文献   

6.
When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. F?rster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.  相似文献   

7.
I Feldman  D Young  R McGuire 《Biopolymers》1975,14(2):335-351
The fluorescence parameters, lifetime, relative quantum yield, maximum and mean wavelength, half-width, and polarization, of bovine serum albumin (BSA) were measured at 15°C in aqueous solutions containing varying concentrations of different chemical perturbants, glycerol, Cu2+ ions, guanidine hydrochloride, and urea. By considering a quenching mechanism as being either dynamic or static, depending upon whether the quenching is or is not accompanied by a change in the fluorescence lifetime, we were able to correlate the changes produced in the various fluorescence parameters by the different chemical perturbants with changes in macromolecular structure as the concentration of perturbant was gradually increased. The addition of glycerol and of Cu2+ ions indicated that in aqueous BSA both tryptophan residues are below the surface of the macromolecule, out of contact with solvent water, and, as a consequence, they are statically quenched. “Ultra-Pure” guanidine hydrochloride at 2.4 M or more caused a drastic conformation change, which resulted in the emergence of a visible tyrosine peak at 304 nm in the BSA fluorescence spectrum when either 260- or 270-nm excitation was employed. With the same excitation, the enhancement of BSA tyrosine fluorescence by 6–8 M ultra-pure urea produced only a shoulder near 304 nm in the BSA fluorescence spectrum. We have introduced the use of a new relative quantum yield for protein fluorescence, q′, referenced to the quantum yield of unquenched free tryptophan, which eliminates the quenching action of water from the reference.  相似文献   

8.
Steady-state and time-resolved fluorescence properties of the single tyrosyl residue in oxytocin and two oxytocin derivatives at pH 3 are presented. The decay kinetics of the tyrosyl residue are complex for each compound. By use of a linked-function analysis, the fluorescence kinetics can be explained by a ground-state rotamer model. The linked function assumes that the preexponential weighting factors (amplitudes) of the fluorescence decay constants have the same relative relationship as the 1H NMR determined phenol side-chain rotamer populations. According to this model, the static quenching of the oxytocin fluorescence can be attributed to an interaction between one specific rotamer population of the tyrosine ring and the internal disulfide bridge.  相似文献   

9.
We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.  相似文献   

10.
Spectroscopic studies of interaction of chlorobenzylidine with DNA   总被引:5,自引:0,他引:5  
Zhong W  Yu JS  Huang W  Ni K  Liang Y 《Biopolymers》2001,62(6):315-323
Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 x 10(5) M(-1) and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of -143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern-Volmer quenching constant obtained from the linear quenching plot is 1.6 x 10(4) M(-1) at 25 degrees C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 +/- 0.4 ns.  相似文献   

11.
The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.  相似文献   

12.
The steady-state and time-resolved fluorescence spectroscopy was applied to determine the influence of an alkyl substituent(s) (methyl or ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, or t-butyl) on amide nitrogen atom on photophysical properties of tyrosine and N-acetyltyrosine amides in water. Generally, the amide group strongly quenches the fluorescence of tyrosine, however, the size and number of substituents on amide nitrogen atom modify the quenching process only in small degree. The fluorescence intensity decays of all amides studied are bi-exponential. The contribution of both components (alphai) to the fluorescence decay undergoes irregular change. An introduction of alkyl substituent on amide nitrogen atom causes an increase of the fluorescence lifetime of tyrosine derivative compared to the unsubstituted amide for both N-acetyltyrosine and tyrosine with the protonated amino group. Calculated, basing on the fluorescence quantum yield (QY) and average lifetime, the radiative rate constants (kf) are similar, which indicates that the substituent(s) does not have substantial influence on radiative process of the deactivation of the excited state of the phenol chromophore for all compounds studied regardless the amino group status as well as the number and type of substituent (linear or branched). The comparison of the ground-state rotamer populations of tyrosine amides and N-acetyltyrosine amides with different alkyl substituent on amide nitrogen atom obtained from 1H NMR with the value of pre-exponential factors indicates that not the rotamer populations, but specific hydration of a whole molecule of the amino acid including chromophore and amino acid moiety, seems to be the main reason of the heterogenous fluorescence intensity decay of tyrosine derivatives.  相似文献   

13.
The fluorescence decay of tryptophan is a sensitive indicator of its local environment within a peptide or protein. We describe the use of frequency domain fluorescence spectroscopy to determine the conformational and environmental changes associated with the interaction of single tryptophan amphipathic peptides with a phospholipid surface. The five 18-residue peptides studied are based on a class A amphipathic peptide known to associate with lipid bilayers. The peptides contain a single tryptophan located at positions 2, 3, 7, 12, or 14 in the sequence. In aqueous solution, the peptides are unstructured and a triple-exponential function is required to fit the decay data. Association of the peptides with small unilamellar vesicles composed of egg phosphatidylcholine reduces the complexity of the fluorescence decays to a double exponential function, with a reduced dependence of the preexponential amplitude on peptide sequence. The data are interpreted in terms of a rotamer model in which the modality and relative proportions of the lifetime components are related to the population distribution of tryptophan chi1 rotamers about the Calpha-Cbeta bond. Peptide secondary structure and the disposition of the tryptophan residue relative to the lipid and aqueous phases in the peptide-lipid complex affect the local environment of tryptophan and influence the distribution of side-chain rotamers. The results show that measurement of the temporal decay of tryptophan emission provides a useful adjunct to other biophysical techniques for investigating peptide-lipid and protein-membrane interactions.  相似文献   

14.
2-Aminopurine (2AP) is an analogue of adenine that has been utilized widely as a fluorescence probe of protein-induced local conformational changes in DNA. Within a DNA strand, this fluorophore demonstrates characteristic decreases in quantum yield and emission decay lifetime that vary sensitively with base sequence, temperature, and helix conformation but that are accompanied by only small changes in emission wavelength. However, the molecular interactions that give rise to these spectroscopic changes have not been established. To develop a molecular model for interpreting the fluorescence measurements, we have investigated the effects of environmental polarity, hydrogen bonding, and the purine and pyrimidine bases of DNA on the emission energy, quantum yield, and intensity decay kinetics of 2AP in simple model systems. The effects of environmental polarity were examined in a series of solvents of varying dielectric constant, and hydrogen bonding was investigated in binary mixtures of water with 1,4-dioxane or N,N-dimethylformamide (DMF). The effects of the purine and pyrimidine bases were studied by titrating 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine (rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosphates ATP and GTP in neutral aqueous solution. The nucleosides and NTPs each quench the fluorescence of d2AP by a combination of static (affecting only the quantum yield) and dynamic (affecting both the quantum yield and the lifetime, proportionately) mechanisms. The peak wavelength and shape of the emission spectrum are not altered by either of these effects. The static quenching is saturable and has half-maximal effect at approximately 20 mM nucleoside or NTP, consistent with an aromatic stacking interaction. The rate constant for dynamic quenching is near the diffusion limit for collisional interaction (k(q) approximately 2 x 10(9) M(-1) s(-1)). Neither of these effects varies significantly between the various nucleosides and NTPs studied. In contrast, hydrogen bonding with water was observed to have a negligible effect on the emission wavelength, fluorescence quantum yield, or lifetime of 2AP in either dioxane or DMF. In nonpolar solvents, the fluorescence lifetime and quantum yield decrease dramatically, accompanied by significant shifts in the emission spectrum to shorter wavelengths. However, these effects of polarity do not coincide with the observed emission wavelength-independent quenching of 2AP fluorescence in DNA. Therefore, we conclude that the fluorescence quenching of 2AP in DNA arises from base stacking and collisions with neighboring bases only but is insensitive to base-pairing or other hydrogen bonding interactions. These results implicate both structural and dynamic properties of DNA in quenching of 2AP and constitute a simple model within which the fluorescence changes induced by protein-DNA binding or other perturbations may be interpreted.  相似文献   

15.
The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.  相似文献   

16.
Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.  相似文献   

17.
The fluorescence lifetime of the single tryptophan in whiting parvalbumin has been measured by time-correlated single-photon counting. In the presence of saturating calcium, greater than 2 mol/mol of protein, the decay of fluorescence is accurately single exponential with a lifetime of 4.6 ns (0.1 M KCl, 20 mM borate, 1 mM dithiothreitol, 20 degrees C, pH 9). Upon complete removal of calcium from parvalbumin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid the emission decay becomes biphasic, and a second more rapid decay process with a lifetime of 1.3 ns comprising approximately 18% of the fluorescence emission at 350 nm is observed. The fluorescence emission of the calcium-saturated form is not measurably quenched by iodide. In contrast, upon complete removal of calcium, the fluorescence is completely quenchable as shown by extrapolation of the data to infinite iodide concentration. These results indicate that there is a large increase in the accessibility of the tryptophan residue in the protein to solvent upon removal of calcium. Stern-Volmer plots of the quenching data are nonlinear and indicate that there is more than one quenchable conformation of the calcium-free protein. The lifetime and quenching results are consistent with the presence of significant concentrations of only two stoichiometric species, apoparvalbumin and parvalbumin--Ca2, at partial occupancy of the calcium binding sites.  相似文献   

18.
U P Andley  B A Clark 《Biochemistry》1988,27(2):810-820
Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.  相似文献   

19.
Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM.  相似文献   

20.
Renfrew PD  Butterfoss GL  Kuhlman B 《Proteins》2008,71(4):1637-1646
Amino acid side chains adopt a discrete set of favorable conformations typically referred to as rotamers. The relative energies of rotamers partially determine which side chain conformations are more often observed in protein structures and accurate estimates of these energies are important for predicting protein structure and designing new proteins. Protein modelers typically calculate side chain rotamer energies by using molecular mechanics (MM) potentials or by converting rotamer probabilities from the protein database (PDB) into relative free energies. One limitation of the knowledge‐based energies is that rotamer preferences observed in the PDB can reflect internal side chain energies as well as longer‐range interactions with the rest of the protein. Here, we test an alternative approach for calculating rotamer energies. We use three different quantum mechanics (QM) methods (second order Møller‐Plesset (MP2), density functional theory (DFT) energy calculation using the B3LYP functional, and Hartree‐Fock) to calculate the energy of amino acid rotamers in a dipeptide model system, and then use these pre‐calculated values in side chain placement simulations. Energies were calculated for over 36,000 different conformations of leucine, isoleucine, and valine dipeptides with backbone torsion angles from the helical and strand regions of the Ramachandran plot. In a subset of cases these energies differ significantly from those calculated with standard molecular mechanics potentials or those derived from PDB statistics. We find that in these cases the energies from the QM methods result in more accurate placement of amino acid side chains in structure prediction tests. Proteins 2008. © 2007 Wiley‐Liss, Inc.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号