Folding energy landscape of cytochrome cb562
Folding energy landscape of cytochrome cb562
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DOI：
10.1073/pnas.0902562106
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发表时间：
2009-05-12
影响因子：
11.1
通讯作者：
Winkler, Jay R.
中科院分区：
文献类型：
--
作者：
Kimura, Tetsunari;Lee, Jennifer C.;Winkler, Jay R.
细胞色素cb（562）是大肠杆菌四螺旋束b型血红素蛋白的变体，其中卟啉辅基共价连接到靠近螺旋4末端的多肽。其他实验室的研究表明，脱辅基蛋白折叠迅速，没有形成中间体，而全蛋白失去血红素之前，天然结构可以获得。使用超快连续流动混合器（150 μ s死区时间）触发的细胞色素cb（562）重折叠的时间分辨荧光能量转移（TRFET）测量表明，血红素与多肽的连接不会干扰天然结构的快速形成。TRFET数据的分析产生分布的色氨酸-59-血红素距离的蛋白质之前，期间，和之后的重折叠。这些分布的时刻和时间演变的表征提供了令人信服的证据，不涉及显着的中间体群体的重折叠机制。这些观察结果表明，细胞色素B（562）折叠能量景观受到最小程度的阻碍，并且能够容忍引入实质性的扰动（即，血红素辅基）而不形成深的错误折叠陷阱。
Cytochrome cb(562) is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome cb(562) refolding triggered using an ultra-fast continuous-flow mixer (150 mu s dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59-heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome b(562) folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps.