Department of Chemistry, Georgetown University, Washington, DC, USA.
Department of Chemistry & Chemical Biology, Cornell University, Ithaca, NY, USA.
Nat Commun. 2023 Feb 24;14(1):1057. doi: 10.1038/s41467-023-36701-y.
The link between cofactor binding and protein activity is well-established. However, how cofactor interactions modulate folding of large proteins remains unknown. We use optical tweezers, clustering and global fitting to dissect the folding mechanism of Drosophila cryptochrome (dCRY), a 542-residue protein that binds FAD, one of the most chemically and structurally complex cofactors in nature. We show that the first dCRY parts to fold are independent of FAD, but later steps are FAD-driven as the remaining polypeptide folds around the cofactor. FAD binds to largely unfolded intermediates, yet with association kinetics above the diffusion-limit. Interestingly, not all FAD moieties are required for folding: whereas the isoalloxazine ring linked to ribitol and one phosphate is sufficient to drive complete folding, the adenosine ring with phosphates only leads to partial folding. Lastly, we propose a dCRY folding model where regions that undergo conformational transitions during signal transduction are the last to fold.
辅因子结合与蛋白质活性之间的联系已得到充分证实。然而，辅因子相互作用如何调节大型蛋白质的折叠仍然未知。我们使用光镊、聚类和全局拟合来剖析果蝇隐花色素（dCRY）的折叠机制，dCRY 是一种 542 个残基的蛋白质，它结合 FAD，这是自然界中化学和结构最复杂的辅因子之一。我们表明，dCRY 最先折叠的部分不依赖于 FAD，但后来的步骤是由 FAD 驱动的，因为剩余的多肽围绕辅因子折叠。FAD 结合于大部分展开的中间体，但结合动力学高于扩散限制。有趣的是，并非所有的 FAD 部分都需要折叠：虽然与核醇和一个磷酸盐相连的异咯嗪环足以驱动完全折叠，但带有磷酸盐的腺嘌呤环只能导致部分折叠。最后，我们提出了一个 dCRY 折叠模型，其中在信号转导过程中发生构象转变的区域是最后折叠的。
