Sborgi Lorenzo, Verma Abhinav, Sadqi Mourad, de Alba Eva, Muñoz Victor
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Methods Mol Biol. 2013;932:205-18. doi: 10.1007/978-1-62703-065-6_13.
The study of protein folding has been conventionally hampered by the assumption that all single-domain proteins fold by an all-or-none process (two-state folding) that makes it impossible to resolve folding mechanisms experimentally. Here we describe an experimental method for the thermodynamic analysis of protein folding at atomic resolution using nuclear magnetic resonance (NMR). The method is specifically developed for the study of small proteins that fold autonomously into basic supersecondary structure motifs, and that do so in the sub-millisecond timescale (folding archetypes). From the NMR experiments we obtain hundreds of atomic unfolding curves that are subsequently analyzed leading to the determination of the characteristic network of folding interactions. The application of this approach to a comprehensive catalog of elementary folding archetypes holds the promise of becoming the first experimental approach capable of unraveling the basic rules connecting protein structure and folding mechanism.
传统上，蛋白质折叠研究受到一种假设的阻碍，即所有单结构域蛋白质都通过全或无过程（两态折叠）进行折叠，这使得通过实验解析折叠机制变得不可能。在此，我们描述了一种利用核磁共振（NMR）在原子分辨率下对蛋白质折叠进行热力学分析的实验方法。该方法专门用于研究能自主折叠成基本超二级结构基序且在亚毫秒时间尺度内完成折叠的小蛋白质（折叠原型）。通过核磁共振实验，我们获得了数百条原子解折叠曲线，随后对其进行分析，从而确定折叠相互作用的特征网络。将这种方法应用于基本折叠原型的综合目录，有望成为第一种能够揭示连接蛋白质结构和折叠机制基本规则的实验方法。
