Habich Markus, Riemer Jan
Institute for Biochemistry, University of Cologne, Zuelpicher Str 47a, 50674, Cologne, Germany.
Methods Mol Biol. 2017;1567:105-138. doi: 10.1007/978-1-4939-6824-4_8.
Import, folding, and activity regulation of mitochondrial proteins are important for mitochondrial function. Cysteine residues play crucial roles in these processes as their thiol groups can undergo (reversible) oxidation reactions. For example, during import of many intermembrane space (IMS) proteins, cysteine oxidation drives protein folding and translocation over the outer membrane. Mature mitochondrial proteins can undergo changes in the redox state of specific cysteine residues, for example, as part of their enzymatic reaction cycle or as adaptations to changes of the local redox environment which might influence their activity. Here we describe methods to study changes in cysteine residue redox states in intact cells. These approaches allow to monitor oxidation-driven protein import as well as changes of cysteine redox states in mature proteins during oxidative stress or during the reaction cycle of thiol-dependent enzymes like oxidoreductases.
线粒体蛋白的导入、折叠及活性调节对于线粒体功能至关重要。半胱氨酸残基在这些过程中发挥着关键作用，因为其巯基可发生（可逆的）氧化反应。例如，在许多膜间隙（IMS）蛋白的导入过程中，半胱氨酸氧化驱动蛋白折叠及通过外膜的转运。成熟的线粒体蛋白可经历特定半胱氨酸残基氧化还原状态的变化，例如，作为其酶促反应循环的一部分，或作为对可能影响其活性的局部氧化还原环境变化的适应性反应。在此，我们描述了研究完整细胞中半胱氨酸残基氧化还原状态变化的方法。这些方法可用于监测氧化驱动的蛋白导入，以及在氧化应激期间或在氧化还原酶等硫醇依赖性酶反应循环期间成熟蛋白中半胱氨酸氧化还原状态的变化。
