Department of Pharmacy, Ludwig-Maximilians-University, Butenandtstr. 5, 81377 Munich, Germany.
Coriolis Pharma Research GmbH, Fraunhoferstr. 18 b, 82152 Martinsried, Germany.
Mol Pharm. 2020 Jul 6;17(7):2638-2647. doi: 10.1021/acs.molpharmaceut.0c00330. Epub 2020 May 27.
Determining the temperature at which the thermal unfolding of a protein starts becoming irreversible is relevant for many areas of protein research. Until now, published methods cannot determine, within a reasonable time frame and with moderate sample consumption, the exposure temperature that starts causing irreversible protein unfolding. We present modulated scanning fluorimetry (MSF) and share a software (MSF Analyzer), which can be used to derive nonreversibility curves of thermal protein unfolding from a series of incremental temperature cycles performed on only 10 μL samples, consuming as low as a few micrograms of protein. Further processing of the data can yield the onset temperature that starts causing nonreversible protein unfolding. The MSF method is based on the hardware of the already existing nanoDSF technology and can be applied to dozens of samples simultaneously. Here, we use MSF to study how solution pH affects the reversibility of thermal protein unfolding of several model proteins to show that the nonreversibility onset temperature () is a unique biophysical parameter, providing orthogonal information from thermal protein denaturation data and insights into the validity of thermal unfolding analysis in the context of equilibrium thermodynamics. We also show that MSF can be used to study enzyme stability after exposure to high temperatures. Besides, we demonstrate that protein thermal unfolding and nonreversibility can be affected in different ways upon modifications like PEG-ylation or labeling with fluorescent dyes. Finally, we show that MSF can be used to study the effect of various protein interactions on thermal protein unfolding reversibility. With the diverse examples in this work, we reveal how MSF can provide orthogonal information from thermal denaturation experiments that can bring benefits to various areas of protein research. The MSF Analyzer software is available at https://github.com/CoriolisPharmaResearch/MSFAnalyser.
确定蛋白质热变性开始变得不可逆的温度与蛋白质研究的许多领域都相关。到目前为止，已发表的方法无法在合理的时间内并以适度的样品消耗来确定导致蛋白质不可逆变性的暴露温度。我们提出了调制扫描荧光法（MSF）并共享了一款软件（MSF Analyzer），该软件可用于从仅对 10μL 样品进行的一系列增量温度循环中得出热蛋白质变性的不可逆性曲线，样品消耗低至几微克蛋白质。进一步处理数据可以得出开始导致蛋白质不可逆变性的起始温度。MSF 方法基于现有的 nanoDSF 技术的硬件，可以同时应用于数十个样品。在这里，我们使用 MSF 研究溶液 pH 值如何影响几种模型蛋白质的热变性的可逆性，以表明不可逆性起始温度（）是一个独特的生物物理参数，它提供了与热变性数据不同的信息，并深入了解了在平衡热力学背景下热变性分析的有效性。我们还表明，MSF 可用于研究在高温下暴露后酶的稳定性。此外，我们证明 MSF 可用于研究蛋白质热变性和不可逆性在经过聚乙二醇化或用荧光染料标记等修饰后以不同的方式受到影响。最后，我们表明 MSF 可用于研究各种蛋白质相互作用对热变性可逆性的影响。通过本工作中的各种示例，我们揭示了 MSF 如何提供与热变性实验不同的正交信息，这将为蛋白质研究的各个领域带来益处。MSF Analyzer 软件可在 https://github.com/CoriolisPharmaResearch/MSFAnalyser 上获取。
