Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.
Nanoscale. 2020 Nov 19;12(44):22564-22573. doi: 10.1039/d0nr06275h.
Metalloproteins play important roles in a wide range of biological processes. The folding process of metalloproteins is complex due to the synergistic effects of the folding of their polypeptide chains and the incorporation of metal cofactors. The folding mechanism of the simplest iron-sulfur protein rubredoxin, which contains one ferric ion coordinated by four cysteinyl sulfurs, is revealed using optical tweezers for the first time. The folding of the rubredoxin polypeptide chain is rapid and robust, while the reconstitution of the iron-sulfur center is greatly dependent upon the coordination state of the ferric ion on the unfolded polypeptide chain. If the ferric ion is coordinated by two neighboring cysteines, rubredoxin can readily fold with the iron-sulfur center fully reconstituted. However, if the ferric ion is only mono-coordinated, rubredoxin can fold but the iron-sulfur center is not reconstituted. Our results suggested that the folding of holo-rubredoxin follows a novel binding-folding-reconstitution mechanism, which is distinct from the folding mechanisms proposed for the folding of metalloproteins. Our study highlights the critical importance of the two-coordinate ferric site in the folding of holo-rubredoxin, which may have some important implications to our understanding of the folding mechanism of more complex metalloproteins in vivo.
金属蛋白在广泛的生物过程中发挥着重要作用。由于其多肽链折叠和金属辅因子掺入的协同作用，金属蛋白的折叠过程较为复杂。本文首次使用光学镊子揭示了最简单的含一个铁离子（由四个半胱氨酸残基配位）的铁硫蛋白 rubredoxin 的折叠机制。rubredoxin 多肽链的折叠迅速且稳健，而铁硫中心的重新形成则极大地依赖于展开多肽链上铁离子的配位状态。如果铁离子由两个相邻的半胱氨酸配位，则 rubredoxin 可以快速折叠并完全重新形成铁硫中心。但是，如果铁离子仅单配位，则 rubredoxin 可以折叠但铁硫中心未重新形成。我们的结果表明，全同型 rubredoxin 的折叠遵循一种新颖的结合-折叠-重新形成机制，与提出的金属蛋白折叠机制不同。我们的研究强调了二配位铁位点在全同型 rubredoxin 折叠中的关键作用，这可能对我们理解体内更复杂金属蛋白的折叠机制具有重要意义。
