Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17777-82. doi: 10.1073/pnas.1201799109. Epub 2012 May 30.
Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module--the FF domain--with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1 · 10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.
蛋白质折叠及其在折叠途径中形成的中间产物的研究为 unfolded ensemble 形成功能性 native conformation 的过程提供了有价值的见解。然而，由于折叠途径中的中间产物可以作为聚集的起始点（涉及多种疾病），因此它们的特征更为重要。由于中间产物的种群往往较低且寿命较短，因此确定它们在折叠、错误折叠和聚集中的作用仍然是一个主要挑战。我们最近使用 NMR 弛豫分散方法和计算技术，以 2-3%的平衡种群和毫秒级的寿命（25°C）确定了小蛋白模块--FF 结构域--的折叠中间产物的原子分辨率结构。基于该结构，设计了一种变体 FF 结构域，其中通过去除天然结构中的羧基末端螺旋来选择性地破坏天然状态，从而产生中间状态的高种群结构模拟物。在这里，我们通过对设计的模拟物的溶液 NMR 研究表明，该模拟物形成了对应于单体和二聚体（K(d) = 0.2 mM）形式的不同构象。这些构象在秒尺度上交换，单体缔合速率为 1.1 · 10(4) M(-1) s(-1)，负责二聚化的区域定位于 FF 结构域的氨基末端残基。这项研究确立了 FF 结构域中间产物作为折叠和错误折叠途径中的核心参与者，并说明了不完全折叠如何导致高级结构的形成。
