天然同位素丰度野生型酵母细胞色素c构象变化的核磁共振检测

Conformational Change of Wild Type Cytochrome c Characterized by NMR Spectroscopy at Natural Isotropic Abundance

细胞色素c是一个重要的多功能蛋白,它在呼吸链及细胞凋亡中均发挥着重要作用.细胞色素c的构象变化与其功能密切相关,表征细胞色素c的构象变化对于明确其相关功能的分子机制至关重要.核磁共振(NMR)是近原位环境下表征蛋白构象的重要工具,但通常需要使用13C、15N等同位素.由于野生型细胞色素c存在翻译后修饰,故其同位素标记十分困难.本文尝试使用1H-13C HSQC技术来表征提纯后的天然同位素丰度的野生型细胞色素c的构象变化.结果显示该方法能在2 h内检测到大多数甲基的信号,且化学位移变化明显的甲基与其构象变化一致.这表明该方法有助于研究天然丰度或翻译后修饰蛋白的构象变化.

Cytochrome c is an important multifunctional protein,which plays important roles in the respiratory chain and cell apoptosis.Characterization of conformational changes of cytochrome c is essential to elucidate the molecular mechanism underlying its functions.Isotope-labeled proteins are usually needed for nuclear magnetic resonance(NMR)-based protein structure elucidation.However,post-translational modification of cytochrome c makes it difficult to be labelled by isotopes.In this work,1H-13C HSQC NMR experiment was used to characterize the conformational changes of naturally purified wild-type cytochrome c in the redox process,focusing on the side chain methyl groups.It was observed that this method could detect the signals of most methyl groups within 2 h,and the methyl groups detected with apparent chemical shift change were coincide with its conformation change.The results indicated that it is possible to use the method proposed to study the conformational changes of proteins at natural abundance or post-translational modified proteins.