Department of Chemistry, University of Leuven, Leuven 3001, Belgium.
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11221, Taiwan.
Sci Adv. 2015 Dec 11;1(11):e1500905. doi: 10.1126/sciadv.1500905. eCollection 2015 Dec.
The emergence of an enzymatic function can reveal functional insights and allows the engineering of biological systems with enhanced properties. We engineered an alpha hemolysin nanopore to function as GroES, a protein that, in complex with GroEL, forms a two-stroke protein-folding nanomachine. The transmembrane co-chaperonin was prepared by recombination of GroES functional elements with the nanopore, suggesting that emergent functions in molecular machines can be added bottom-up by incorporating modular elements into preexisting protein scaffolds. The binding of a single-ring version of GroEL to individual GroES nanopores prompted large changes to the unitary nanopore current, most likely reflecting the allosteric transitions of the chaperonin apical domains. One of the GroEL-induced current levels showed fast fluctuations (<1 ms), a characteristic that might be instrumental for efficient substrate encapsulation or folding. In the presence of unfolded proteins, the pattern of current transitions changed, suggesting a possible mechanism in which the free energy of adenosine triphosphate binding and hydrolysis is expended only when substrate proteins are occupied.
酶功能的出现可以揭示功能见解，并允许具有增强性能的生物系统的工程设计。我们设计了一种α溶血素纳米孔作为 GroES，这种蛋白质与 GroEL 结合形成一个两冲程的蛋白质折叠纳米机器。跨膜共伴侣是通过将 GroES 功能元件与纳米孔重组制备的，这表明可以通过将模块化元件纳入预先存在的蛋白质支架中，从底部向上添加分子机器中的新兴功能。单个环版本的 GroEL 与单个 GroES 纳米孔的结合促使单位纳米孔电流发生了很大的变化，这很可能反映了伴侣蛋白顶端结构域的变构跃迁。GroEL 诱导的电流水平之一显示出快速波动（<1 ms），这一特征可能对有效底物包封或折叠很重要。在未折叠蛋白质存在的情况下，电流转换模式发生变化，这表明可能存在一种机制，即只有当底物蛋白被占据时，三磷酸腺苷结合和水解的自由能才会被消耗。
