Center for Structural Biology, Vanderbilt University, Nashville, Tennessee; Department of Chemistry, Vanderbilt University, Nashville, Tennessee.
Center for Structural Biology, Vanderbilt University, Nashville, Tennessee; Department of Chemistry, Vanderbilt University, Nashville, Tennessee; Institute of Drug Discovery, Faculty of Medicine, University of Leipzig, Leipzig, Germany.
Biophys J. 2024 Jan 2;123(1):68-79. doi: 10.1016/j.bpj.2023.11.019. Epub 2023 Nov 20.
Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the "dark" nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (T) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.
测量蛋白质热稳定性为研究蛋白质结构与能量关系的生物物理规律提供了有价值的信息。然而，这种测量方法对膜蛋白仍然是一个挑战。在这里，我们引入了一种新的实验系统来评估膜蛋白的热稳定性。该系统利用最近开发的非荧光膜支架蛋白将蛋白质重新组装成纳米盘，并与纳米格式差示扫描荧光法（nanoDSF）相结合。这种方法提供了一种无标记的、直接测量膜蛋白在溶液中展开时固有色氨酸荧光的方法，而不会受到“暗”纳米盘的信号干扰。在这项工作中，我们使用二硫键形成蛋白 B（DsbB）作为测试膜蛋白来演示该方法的应用。用 1,2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱（DMPC）和 1,2-二肉豆蔻酰-sn-甘油-3-磷酸甘油（DMPG）脂质负载的暗纳米盘中重新组装的 DsbB 的 nanoDSF 测量显示出复杂的双相热展开模式，具有较小的展开过渡，随后是主要过渡。热变性曲线的拐点揭示了两个不同的展开中点熔融温度（T），分别为 70.5°C 和 77.5°C，与三态展开模型一致。此外，我们表明，残基 C41 和 C130 之间的催化保守二硫键驱动 DsbB 在 DMPC 和 DMPG 纳米盘中展开途径的中间状态。为了扩展该方法的用途，我们评估并比较了 DsbB 在不同脂质环境中的热稳定性。我们将该方法引入为一种新工具，可以用于了解组成和生物物理复杂的脂质环境如何驱动膜蛋白稳定性。
