Bader Arjen N, Visser Nina V, van Amerongen Herbert, Visser Antonie J W G
Laboratories of Biophysics and Biochemistry, Microspectroscopy Centre, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands.
Methods Appl Fluoresc. 2014 Nov 7;2(4):045001. doi: 10.1088/2050-6120/2/4/045001.
The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and exhibit only minor shifts. In this work, we show that phasor approaches can substantially simplify tryptophan fluorescence analysis. To demonstrate this, we re-analyse previously recorded datasets of the denaturant (guanidinium hydrochloride, GuHCl) induced unfolding of a single-tryptophan-containing variant of apoflavodoxin from Azotobacter vinelandii. For three methods-(1) time-resolved fluorescence, (2) time-resolved fluorescence anisotropy and (3) steady-state fluorescence spectroscopy-we show that the phasor analysis can readily identify the presence of a folding intermediate. Moreover, the fractional contributions of protein states at various stages of unfolding and the values of the free energy difference of the unfolding process [Formula: see text] are obtained. The outcomes are compared to the global analysis results published previously.
色氨酸的固有荧光常用于研究蛋白质的结构。色氨酸荧光数据的分析具有挑战性：荧光（各向异性）衰减通常具有多个寿命（相关时间）成分，并且荧光光谱很宽，仅表现出微小的位移。在这项工作中，我们表明相量法可以大大简化色氨酸荧光分析。为了证明这一点，我们重新分析了先前记录的数据集，该数据集是关于来自棕色固氮菌的无黄素黄素氧还蛋白的单色氨酸变体在变性剂（盐酸胍，GuHCl）诱导下的展开情况。对于三种方法——（1）时间分辨荧光、（2）时间分辨荧光各向异性和（3）稳态荧光光谱——我们表明相量分析可以很容易地识别折叠中间体的存在。此外，还获得了蛋白质在展开各个阶段的状态的分数贡献以及展开过程的自由能差的值[公式：见正文]。将结果与先前发表的全局分析结果进行了比较。
