共查询到19条相似文献,搜索用时 171 毫秒
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采用时间相关单光子计数技术, 结合紫外-可见吸收光谱和稳态荧光光谱, 对不同环境下的色氨酸和辅酶还原型烟酰胺腺嘌呤二核苷酸(NADH)之间的共振能量转移荧光动力学进行了研究. 单体色氨酸、 牛血清白蛋白以及乳酸脱氢酶蛋白与NADH之间相互作用的光谱数据表明, 只有存在NADH结合位点的乳酸脱氢酶和NADH之间发生了荧光共振能量转移. 进一步通过加入丙酮酸来阻断乳酸脱氢酶和NADH之间的荧光共振能量转移通道, 时间分辨荧光光谱和衰减相关光谱(DAS)证实, 蛋白结合位点的存在是NADH和色氨酸之间发生荧光共振能量转移的前提条件. DAS揭示了乳酸脱氢酶平均荧光寿命的减小主要是源于色氨酸中7.35 ns的荧光寿命成分与NADH之间的荧光共振能量转移, 同时给出了NADH和色氨酸之间的能量转移效率, 为研究NADH和蛋白之间的相互作用提供了新思路. 相似文献
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测得了长白山白眉蝮蛇毒精氨酸酯酶 1的最适反应的pH范围为 7.0~ 8.0 ,且与酶反应底物对甲苯磺酰-L -精氨酸甲酯 (TAME)的反应无明显的最适应反应温度 .荧光光谱的研究结果表明 :该酶的酪氨酸残基的荧光被色氨酸残基的荧光所掩盖 ;同步荧光光谱结果表明 :当发射波长与激发波长差Δλ分别为 2 0nm和 75nm时 ,精氨酸酯酶 1的荧光光谱分别由酪氨酸 (Tyr)和色氨酸 (Trp)残基所贡献 ,且处于亲水性环境中 ;精氨酸酯酶 1的荧光发射强度受溶液酸度变化的影响 .I- ,Acr和NBS对精氨酸酯酶 1的荧光淬灭结果表明这种酶中含有多个色氨酸残基 ,且处于不同的微环境中。 相似文献
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采用分子对接技术和同步荧光光谱法、红边激发荧光位移法(REES法)及圆二色谱法(CD)共同研究了山柰酚与牛血清白蛋白(BSA)在pH7.40的缓冲溶液中的相互作用。分子对接的结果表明,山柰酚的B环插入到BSA的ⅡA结构域中的疏水腔内,与色氨酸残基(Trp212)的距离为12.96,维系药物与蛋白质的主要作用力为疏水作用。通过荧光光谱法测得二者之间相互作用力主要为疏水性相互作用,结合位点为1,与分子模拟结果一致。同步荧光光谱及REES法的研究表明,发生相互作用的过程中BSA的色氨酸残基处于运动受限的微环境中,而适当增加山柰酚的浓度能够改变色氨酸微环境的流动性,进而对BSA的构象产生一定影响;同时,圆二色谱的定量计算结果也表明,一定浓度的山柰酚与BSA的相互作用引起了α-螺旋含量的显著降低,从11.91%降低到1.67%,对BSA的二级结构产生一定影响。 相似文献
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采用紫外-可见光谱、荧光光谱、同步荧光光谱和圆二色光谱法研究了肌红蛋白与亚硝酸钠配位反应的光谱学性质.结果表明,肌红蛋白与亚硝酸钠配位反应的进行与血红素中心铁原子的价态有关;当肌红蛋白活性中心Fe为三价时,亚硝酸钠主要与蛋白质上的氨基酸发生作用.而当肌红蛋白的活性中心Fe为二价时,亚硝酸钠则与卟啉铁配位,形成亚硝基亚铁肌红蛋白.荧光光谱表明亚硝酸钠的加入改变了肌红蛋白氨基酸残基的微环境,同步荧光光谱数据显示肌红蛋白与亚硝酸钠作用位点更接近于色氨酸残基,同时CD数据也表明肌红蛋白与亚硝酸钠作用后,其α-螺旋含量降低了19.33%,二级结构发生明显变化. 相似文献
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水溶性羟基化单壁碳纳米管与人血清白蛋白之间的相互作用研究 总被引:2,自引:1,他引:1
利用荧光光谱、UV吸收光谱、同步荧光光谱和透射电子显微镜等技术较为系统地研究了水溶性羟基化单壁碳纳米管与人血清白蛋白(HSA)的相互作用. 实验发现, 这种羟基化单壁碳纳米管可以明显猝灭HSA的内源荧光, 且猝灭效应随碳纳米管浓度增大而增强. 同时, HSA对水溶性羟基化单壁碳纳米管起到一定的分散和稳定作用. 同步荧光光谱表明, 二者之间的相互作用可导致HSA的构象发生变化, HSA的色氨酸残基荧光信号发生2 nm的红移, 表明色氨酸残基周围微环境的极性降低. 相似文献
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Stortelder A Hendriks J Buijs JB Bulthuis J Gooijer C van der Vies SM van der Zwan G 《The journal of physical chemistry. B》2006,110(49):25050-25058
The bacteriophage T4 capsid protein gp23 was studied using time-resolved and steady-state fluorescence of the intrinsic protein fluorophore tryptophan. In-vitro gp23 consists mostly of monomers at low temperature but forms hexamers at room temperature. To extend our knowledge of the structure and hexamerization characteristics of gp23, the temperature-dependent fluorescence properties of a tryptophan mutant (W13V) were compared to those of wild-type gp23. The W13V mutation is located in the N-terminal part of the protein, which is cleaved off after prohead formation in the live bacteriophage. Results show that W13 plays a role in the hexamerization process but is not needed to stabilize the hexamer once it is formed. Furthermore, besides the monomer-to-hexamer temperature transition (15-23 degrees C and 12-43 degrees C for wild-type and W13V gp23, respectively), we were able to observe denaturation of the N-terminus in hexameric wild-type gp23 around 40 degrees C. In addition, with the aid of a recently published homology model of gp23, the lifetimes obtained from time-resolved fluorescence measurements could tentatively be assigned to specific tryptophan residues. 相似文献
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Xu J Toptygin D Graver KJ Albertini RA Savtchenko RS Meadow ND Roseman S Callis PR Brand L Knutson JR 《Journal of the American Chemical Society》2006,128(4):1214-1221
The complete time-resolved fluorescence of tryptophan in the proteins monellin and IIA(Glc) has been investigated, using both an upconversion spectrophotofluorometer with 150 fs time resolution and a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. In monellin, the fluorescence decay displays multiexponential character with decay times of 1.2 and 16 ps, and 0.6, 2.2, and 4.2 ns. In contrast, IIA(Glc) exhibited no component between 1.2 ps and 0.1 ns. For monellin, surprisingly, the 16 ps fluorescence component was found to have positive amplitude even at longer wavelengths (e.g., 400 nm). In conjunction with quantum mechanical simulation of tryptophan in monellin, the experimental decay associated spectra (DAS) and time-resolved emission spectra (TRES) indicate that this fluorescence decay time should be ascribed to a highly quenched conformer. Recent models (Peon, J.; et al. Proc. Natl.Acad. Sci. U.S.A. 2002, 99, 10964) invoked exchange-coupled relaxation of protein water to explain the fluorescence decay of monellin. 相似文献
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Camillo A. Ghiron Maurice R. Eftink David A. Engler Salil K. Niyogi 《Photochemistry and photobiology》1992,55(1):29-34
Steady-state and time-resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C-terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub-nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low-temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp-trp), it is concluded that homo-energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady-state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C-terminus segment, which is not an integral part of the three-dimensional structure of the protein. Fluorescence measurements with three site-directed mutants also show very little variation. 相似文献
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Stortelder A Buijs JB Bulthuis J van der Vies SM Gooijer C van der Zwan G 《Journal of photochemistry and photobiology. B, Biology》2005,78(1):53-60
The time-resolved fluorescence properties of the bacteriophage T4 capsid protein gp23 are investigated. The structural characteristics of this protein are largely unknown and can be probed by recording time-resolved and decay-associated fluorescence spectra and intensity decay curves using a 200 ps-gated intensified CCD-camera. Spectral and decay data are recorded simultaneously, which makes data acquisition fast compared to time-correlated single-photon counting. A red-shift of the emission maximum within the first nanosecond of decay is observed, which can be explained by the different decay-associated spectra of fluorescence lifetimes of the protein in combination with dipolar relaxation. In addition, iodide quenching experiments are performed, to study the degree of exposure of the various tryptophan residues. A model for the origin of the observed lifetimes of 0.032 +/- 0.003, 0.39 +/- 0.06, 2.1 +/- 0.1 and 6.8 +/- 0.8 ns is presented: the 32 ps lifetime can be assigned to the emission of a buried tryptophan residue, the 0.4 and 2.1 ns lifetimes to two partly buried residues, and the 6.8 ns lifetime to a single tryptophan outside the bulk of the folded gp23. 相似文献
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S. R. Meech D. V. O'Connor A. J. Roberts D. Phillips 《Photochemistry and photobiology》1981,33(2):159-172
Abstract Two common methods of obtaining nanosecond time-resolved spectra (TRES) are compared. TRES measured directly are distorted owing to convolution of the fluorescence signal with the exciting pulse but can be obtained with ease. Undistorted TRES, constructed from deconvoluted decay curves, suffer from poor spectral resolution, require much experimental and computation time to produce and may not be completely free from distortions. Nevertheless, they must be used for quantitative calculations. It is recommended that the method of obtaining TRES should be determined by the type of information required. 相似文献
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Nakamura R Hamada N Ichida H Tokunaga F Kanematsu Y 《Photochemistry and photobiology》2008,84(4):937-940
The ultrafast photo-induced dynamics of wild-type photoactive yellow protein and its site-directed mutant of E46Q in aqueous solution was studied at room temperature by femtosecond fluorescence spectroscopy using the optical Kerr-gate method. The vibronic structure appears, depending on the excitation photon energy, in the time-resolved fluorescence spectra just after photoexcitation, which winds with time and disappears on a time scale of sub-picoseconds. This result indicates that the wavepacket is localized in the electronic excited state followed by dumped oscillations and broadening, and also that the initial condition of the wavepacket prepared depending on the excitation photon energy affects much the following ultrafast dynamics in the electronic excited state. 相似文献
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The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification. 相似文献
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Akkaladevi Narahari Musti J. Swamy 《Journal of photochemistry and photobiology. B, Biology》2009,97(1):40-47
The exposure and accessibility of the tryptophan residues in the chitooligosaccharide-specific pumpkin (Cucurbita maxima) phloem exudate lectin (PPL) have been investigated by fluorescence spectroscopy. The emission λmax of native PPL, seen at 338 nm was red-shifted to 348 nm upon denaturation by 6 M Gdn.HCl in the presence of 10 mM β-mercaptoethanol, indicating near complete exposure of the tryptophan residues to the aqueous medium, whereas a blue-shift to 335 nm was observed in the presence of saturating concentrations of chitotriose, suggesting that ligand binding leads to a decrease in the solvent exposure of the tryptophan residues. The extent of quenching was maximum with the neutral molecule, acrylamide whereas the ionic species, iodide and Cs+ led to significantly lower quenching, which could be attributed to the presence of charged amino acid residues in close proximity to some of the tryptophan residues. The Stern–Volmer plot for acrylamide was linear for native PPL and upon ligand binding, but became upward curving upon denaturation, indicating that the quenching occurs via a combination of static and dynamic mechanisms. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, for native protein, in the presence of ligand and upon denaturation. In each case both lifetimes systematically decreased with increasing acrylamide concentrations, indicating that quenching occurs predominantly via a dynamic process. 相似文献