Sadqi Mourad, Fushman David, Muñoz Victor
Department of Chemistry and Biochemistry, and Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742, USA.
Nature. 2006 Jul 20;442(7100):317-21. doi: 10.1038/nature04859. Epub 2006 Jun 14.
Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.
蛋白质折叠是一个本质上复杂的过程，涉及稳定天然三维结构的弱相互作用复杂网络的协调。在传统范式中，简单的蛋白质结构被认为是以一种实验无法触及的全或无过程进行折叠的。因此，现有的实验方法只能间接探测折叠机制。一种广泛使用的方法是根据天然蛋白质的结构来解释由工程突变引起的蛋白质稳定性和/或折叠动力学的变化。除了在将能量学与结构联系起来方面存在局限性外，突变方法还存在重大的实验不确定性，并且无法绘制复杂的相互作用网络。相比之下，分析理论预测折叠存在小的障碍以及存在下坡折叠的可能性。近年来，这些理论预测已通过实验得到证实，包括对全局下坡折叠的观察。然而，一个关键的遗留问题是下坡折叠是否真的能导致对蛋白质折叠过程进行高分辨率分析。在这里，我们使用核磁共振（NMR）表明，来自大肠杆菌的下坡蛋白BBL从一个确定的三维结构开始逐个原子地展开。在总共204个质子中，对158个主链和侧链质子的热展开数据提供了折叠过程中结构事件的详细视图。这一视图证实了折叠的统计性质，并揭示了氢键、疏水力、主链构象和侧链熵之间的相互作用。从这些数据中，我们还获得了该蛋白质中相互作用网络的图谱，揭示了折叠协同性的来源。我们的方法可以扩展到其他具有微小障碍（小于3RT）的蛋白质，为蛋白质折叠研究提供了一种新工具。
