Abstract：
Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.
摘要：
由于其固有的复杂性，评估膜蛋白稳定性是蛋白质科学的主要挑战之一。这使得传统生物物理工具的应用复杂化。在这项工作中,十二烷基硫酸钠诱导的AfCopA变性，一种来自古细菌的Cu（I）转运ATP酶，使用组合的无模型光谱相量分析和模型相关的热力学分析进行了探索。色氨酸和1-苯胺基-萘-8-磺酸盐荧光强度降低，光谱相量空间中的位移，并且该洗涤剂可逆地诱导了ATPase活性的丧失。从SDS诱导的变性状态重折叠产生活性酶,其在功能和光谱上与蛋白质的天然状态没有区别。Trp光谱的相量分析使我们能够识别SDS诱导的AfCopA变性中的两个中间状态，主成分分析进一步支持了这一结果。相比之下,传统的热力学分析只检测到一个中间状态，包括第二个导致过度参数化。此外,ANS荧光光谱分析检测到了一种在疏水性跨膜表面的蛋白质的中间和渐变水平。根据这些证据,提出了一种在膜状环境中获取AfCopA天然结构的模型。
