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1.
2.
Frequency domain measurements of the fluorescence lifetime of ribonuclease T1. 总被引:2,自引:1,他引:1
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Using multifrequency phase/modulation fluorometry, we have studied the fluorescence decay of the single tryptophan residue of ribonuclease T1 (RNase T1). At neutral pH (7.4) we find that the decay is a double exponential (tau 1 = 3.74 ns, tau 2 = 1.06 ns, f1 = 0.945), in agreement with results from pulsed fluorometry. At pH 5.5 the decay is well described by a single decay time (tau = 3.8 ns). Alternatively, we have fitted the frequency domain data by a distribution of lifetimes. Temperature dependence studies were performed. If analyzed via a double exponential model, the activation energy for the inverse of the short lifetime component (at pH 7.4) is found to be 3.6 kcal/mol, as compared with a value of 1.0 kcal/mol for the activation energy of the inverse of the long lifetime component. If analyzed via the distribution model, the width of the distribution is found to increase at higher temperature. We have also repeated, using lifetime measurements, the temperature dependence of the acrylamide quenching of the fluorescence of RNase T1 at pH 5.5. We find an activation energy of 8 kcal/mol for acrylamide quenching, in agreement with our earlier report. 相似文献
3.
Fluorescence lifetime studies with staphylococcal nuclease and its site-directed mutant. Test of the hypothesis that proline isomerism is the basis for nonexponential decays. 总被引:1,自引:0,他引:1
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Using frequency domain methods, the fluorescence decay of Trp-140 in staphylococcal nuclease and its site-directed mutant (Pro-117----Gly) has been examined. Based on nuclear magnetic resonance (NMR) studies (Evans, P. A., C. M. Dobson, R. A. Kautz, G. Hatfull, and R. O. Fox. 1987. Nature [Lond.]. 329:266-268), it is believed that nuclease exists in two macroscopic, native conformations and that the slow interconversion of these conformations is controlled by the cis----trans isomerization of Pro-117. The above mutant shows only one native conformation in NMR experiments. To test the hypothesis that the biexponential fluorescence decay of Trp-140 of nuclease can also be related to the existence of these conformational states of the protein, we have compared the decay patterns of the wild type and mutant. Essentially no difference was observed, which indicates that there is some other basis for the nonexponential decay of Trp-140. We have used global nonlinear least squares analysis to link the fit of data at several temperatures. 相似文献
4.
Effect of micelle diameter on tryptophan dynamics in an amphipathic helical peptide in phosphatidylcholine 总被引:1,自引:0,他引:1
The effect of dimyristoylphosphatidylcholine (DMPC) on the conformation and environment of the single tryptophan residue of a model amphipathic helical polypeptide has been investigated by fluorescence quenching with a water-soluble, neutral quencher (acrylamide) and multiple-frequency phase fluorometry. The peptide H-Ser-Ser-Ala-Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Ly s-Glu- Ala-Phe-Ser-Ser-Ser-OH [18As; Kanellis, P., Romans, A.Y., Johnson, B.J., Kercret, H., Chiovetti, R., Jr., Allen, T.M., & Segrest, S.P. (1980) J. Biol. Chem. 255, 11464] was synthesized by solid-phase techniques. Peptide was incubated at 26 degrees C with DMPC at various peptide:lipid weight ratios. The diameter of the resulting disk-shaped micelles increases with increasing lipid concentration from 12.0 +/- 0.4 nm at a 1:1 weight ratio of peptide to lipid to a maximum of 48.7 +/- 1.0 nm at a 1:13 ratio. At a weight ratio of 1:5, the average diameter is 22.7 +/- 0.6 nm. Decreasing the peptide:lipid ratio of the micelle resulted in a blue-shift in the fluorescence emission maximum (from 337 nm at 1:1 to 334 nm at 1:5), an increase in the fluorescence lifetime of the tryptophan measured by the phase shift method at 18 MHz (from 3.12 ns at 1:1 to 3.61 ns at 1:5), a decrease in the rate of fluorescence quenching by acrylamide (from 0.87 x 10(9) M-1 s-1 at 1:1 to 0.42 x 10(9) M-1 s-1 at 1:5), and an increase in the activation energy for quenching (from 6.7 kcal/mol at 1:1 to 12.7 kcal/mol at 1:5).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
From measurements of the apparent phase and modulation fluorescence lifetime of liver alcohol dehydrogenase at multiple modulation frequencies (6, 18, and 30 MHz), the individual lifetimes and fractional intensities of Trp-314 and Trp-15 are calculated. Values of tau 314 = 3.6, tau 15 = 7.3, and f314 = 0.56, at 20 degrees C, are found. These values are in general agreement with values previously reported by Ross et al. [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369] using pulse-decay methodology. In ternary complexes formed between the enzyme, NAD+ and either pyrazole or trifluoroethanol, the fluorescence lifetime of Trp-314 is found to be reduced, indicating that the binding of these ligands causes a dynamic quenching of this residue. The lifetime of Trp-314 is decreased more in the trifluoroethanol ternary complex than that with pyrazole. Also, the alkaline quenching transition of alcohol dehydrogenase is found to result in the selective, dynamic quenching of Trp-314. No change in the lifetimes of the two Trp residues is found upon selective removal of the active-site zinc atoms. From studies of the fluorescence anisotropy, r, of the enzyme as a function of added acrylamide (which selectively quenches the surface Trp-15 residue), the steady-state anisotropy of each residue is determined to be r314 = 0.26 and r15 = 0.21. In the ternary complexes the anisotropy of each residue increases slightly.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide. 相似文献
7.
Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins 总被引:3,自引:1,他引:2
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M Eftink 《Biophysical journal》1983,43(3):323-334
Measurements of the anisotropy of protein fluorescence as a function of an added collisional quencher, such as acrylamide, are used to construct Perrin plots. For single tryptophan containing proteins, such plots yield an apparent rotational correlation time for the depolarization process, which, in most cases, is approximately the value expected for Brownian rotation of the entire protein. Apparent limiting fluorescence anisotropy values, which range from 0.20 to 0.32 for the proteins studied, are also obtained from the Perrin plots. The lower values for the limiting anisotropy found for some proteins are interpreted as indicating the existence of relatively rapid, limited (within a cone of angle 0 degrees--30 degrees) motion of the tryptophan side chains that is independent of the overall rotation of the protein. Examples of the use of this fluorescence technique to study protein conformational changes are presented, including the monomer in equilibrium dimer equilibrium of beta-lactoglobulin, the monomer in equilibrium tetramer equilibrium of melittin, the N in equilibrium F transition of human serum albumin, and the induced change in the conformation of cod parvalbumin caused by the removal of Ca+2. Because multitryptophan-containing proteins have certain tryptophans that are accessible to solute quencher and others that are inaccessible, this method can be used to determine the steady state anisotropy of each class of tryptophan residues. 相似文献
8.
9.
Frequency-domain fluorescence spectroscopy was used to investigate the effects of temperature on the intensity and anisotropy decays of the single tryptophan residues of Staphylococcal nuclease A and its nuclease-conA-SG28 mutant. This mutant has the beta-turn forming hexapeptide, Ser-Gly-Asn-Gly-Ser-Pro, substituted for the pentapeptide Tyr-Lys-Gly-Gln-Pro at positions 27-31. The intensity decays were analyzed in terms of a sum of exponentials and with Lorentzian distributions of decay times. The anisotropy decays were analyzed in terms of a sum of exponentials. Both the intensity and anisotropy decay parameters strongly depend on temperature near the thermal transitions of the proteins. Significant differences in the temperature stability of Staphylococcal nuclease and the mutant exist; these proteins show characteristic thermal transition temperatures (Tm) of 51 and 30 degrees C, respectively, at pH 7. The temperature dependence of the intensity decay data are shown to be consistent with a two-state unfolding model. For both proteins, the longer rotational correlation time, due to overall rotational diffusion, decreases dramatically at the transition temperature, and the amplitude of the shorter correlation time increases, indicating increased segmental motions of the single tryptophan residue. The mutant protein appears to have a slightly larger overall rotational correlation time and to show slightly more segmental motion of its Trp than is the case for the wild-type protein. 相似文献
10.
Danilo ML Prado Fabiana B Benatti Ana L de Sá-Pinto Ana P Hayashi Bruno Gualano Rosa MR Pereira Adriana ME Sallum Eloisa Bonfá Clovis A Silva Hamilton Roschel 《Arthritis research & therapy》2013,15(2):R46