Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
J Mol Biol. 2010 Apr 2;397(3):777-88. doi: 10.1016/j.jmb.2010.01.056. Epub 2010 Feb 6.
Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu-->Glu(-)) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.
尽管大多数折叠中间体难以检测到，但它们的特性对于阐明折叠机制至关重要。在这里，我们概述了一种产生部分展开中间体的强大策略：用带电荷的残基（例如 Leu-->Glu(-)）取代埋藏的脂肪族残基，以破坏和展开蛋白质的特定区域。我们将此策略应用于泛素，可逆地捕获其中β5 链展开的折叠中间体。在温和的酸性条件下中和电荷时，中间体重新折叠成类似天然的结构。使用 NMR 和氢交换方法对捕获的中间体进行的表征确定了第二个折叠中间体，并揭示了限速步骤的天然侧的两个主要折叠事件的顺序和自由能。这种通用策略可以与其他方法结合使用，并在蛋白质折叠和其他需要捕获高能状态的反应的研究中具有广泛的应用。
