Cristian Lidia, Zhang Yao
Influmedix, Inc., Radnor, PA, USA.
Methods Mol Biol. 2013;1063:3-18. doi: 10.1007/978-1-62703-583-5_1.
The development of methods for reversibly folding membrane proteins in a two-state manner remains a considerable challenge for studies of membrane protein stability. In recent years, a variety of techniques have been established and studies of membrane protein folding thermodynamics in the native bilayer environments have become feasible. Here we present the thiol-disulfide exchange method, a promising experimental approach for investigating the thermodynamics of transmembrane (TM) helix-helix association in membrane-mimicking environments. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. This experimental method provides a broadly applicable tool for thermodynamic studies of folding, oligomerization, and helix-helix interactions of membrane proteins.
以双态方式可逆折叠膜蛋白的方法的开发，对于膜蛋白稳定性研究而言仍然是一项巨大挑战。近年来，已建立了多种技术，在天然双层环境中研究膜蛋白折叠热力学变得可行。在此，我们介绍硫醇 - 二硫键交换方法，这是一种在膜模拟环境中研究跨膜（TM）螺旋 - 螺旋缔合热力学的有前景的实验方法。该方法涉及在硫醇 - 二硫键缓冲液中可逆氧化还原条件下引发蛋白质的二硫键交联，并在平衡时对交联程度进行定量评估。这种实验方法为膜蛋白的折叠、寡聚化和螺旋 - 螺旋相互作用的热力学研究提供了一种广泛适用的工具。
