Kathuria Sagar V, Day Iain J, Wallace Louise A, Matthews C Robert
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
J Mol Biol. 2008 Oct 3;382(2):467-84. doi: 10.1016/j.jmb.2008.06.054. Epub 2008 Jun 28.
The beta alpha-repeat class of proteins, represented by the (beta alpha)(8) barrel and the alpha/beta/alpha sandwich, are among the most common structural platforms in biology. Previous studies on the folding mechanisms of these motifs have revealed or suggested that the initial event involves the submillisecond formation of a kinetically trapped species that must at least partially unfold before productive folding to the respective native conformation can occur. To test the generality of these observations, CheY, a bacterial response regulator, was subjected to an extensive analysis of its folding reactions. Although earlier studies had proposed the formation of an off-pathway intermediate, the data available were not sufficient to rule out an alternative on-pathway mechanism. A global analysis of single- and double-jump kinetic data, combined with equilibrium unfolding data, was used to show that CheY folds and unfolds through two parallel channels defined by the state of isomerization of a prolyl peptide bond in the active site. Each channel involves a stable, highly structured folding intermediate whose kinetic properties are better described as the properties of an off-pathway species. Both intermediates subsequently flow through the unfolded state ensemble and adopt the native cis-prolyl isomer prior to forming the native state. Initial collapse to off-pathway folding intermediates is a common feature of the folding mechanisms of beta alpha-repeat proteins, perhaps reflecting the favored partitioning to locally determined substructures that cannot directly access the native conformation. Productive folding requires the dissipation of these prematurely folded substructures as a prelude to forming the larger-scale transition state that leads to the native conformation. Results from Gō-modeling studies in the accompanying paper elaborate on the topological frustration in the folding free-energy landscape of CheY.
以（β-α）8桶和α/β/α三明治为代表的β-α重复类蛋白质是生物学中最常见的结构平台之一。先前对这些基序折叠机制的研究已经揭示或表明，初始事件涉及在亚毫秒内形成一个动力学捕获的物种，该物种在有效折叠成各自的天然构象之前必须至少部分展开。为了检验这些观察结果的普遍性，对细菌应答调节蛋白CheY的折叠反应进行了广泛分析。尽管早期研究提出了一种非途径中间体的形成，但现有的数据不足以排除另一种途径上的机制。对单跳和双跳动力学数据以及平衡展开数据进行全局分析，结果表明CheY通过由活性位点中脯氨酰肽键的异构化状态定义的两个平行通道进行折叠和展开。每个通道都涉及一个稳定的、高度结构化的折叠中间体，其动力学性质更适合描述为非途径物种的性质。这两种中间体随后都流经未折叠状态的集合，并在形成天然状态之前采用天然顺式脯氨酰异构体。初始折叠为非途径折叠中间体是β-α重复蛋白折叠机制的一个共同特征，这可能反映了有利于分配到局部确定的亚结构，而这些亚结构无法直接形成天然构象。有效折叠需要消除这些过早折叠亚结构，作为形成导致天然构象的更大规模过渡态的前奏。随附论文中的Gō模型研究结果详细阐述了CheY折叠自由能景观中的拓扑挫折。
