Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry, Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Nat Commun. 2016 Jul 15;7:12163. doi: 10.1038/ncomms12163.
Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1(Ubix), the cofactor confers structural stability to the enzyme. IM-MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM-MS data.
Fdc1 是一种脱羧酶，其活性需要新型的异戊二烯化 FMN 辅因子。在这里，我们将其用作示例系统，展示天然的自上而下和自下而上的质谱如何测量辅因子结合对蛋白质结构的影响。对于 Fdc1(Ubix)，辅因子赋予酶结构稳定性。IM-MS 表明全酶存在于四个密切相关的构象家族中，在无辅因子的情况下，其种群与apo 形式不同；在有辅因子的情况下，两个较小的家族更具代表性，而在没有辅因子的情况下则较少。这些发现得到 MD 模拟的支持，表明 apo 形式的结构更开放。HDX-MS 表明，虽然主要的结构变化发生在靠近辅因子结合位点的地方，但在结合辅因子时，整个蛋白质都发生了重排，这主要归因于变构构象的收紧，与 IM-MS 数据一致。
