Li Meng-Yin, Wang Ya-Qian, Ying Yi-Lun, Long Yi-Tao
State Key Laboratory of Analytical Chemistry for Life Science , School of Chemistry and Chemical Engineering , 210023 , Nanjing , P. R. China . Email:
School of Chemistry and Molecule Engineering , East China University of Science and Technology , 200237 , Shanghai , P. R. China.
Chem Sci. 2019 Sep 23;10(44):10400-10404. doi: 10.1039/c9sc03163d. eCollection 2019 Nov 28.
Flavin adenine dinucleotide (FAD) as a cofactor is involved in numerous important metabolic pathways where the biological function is intrinsically related to its transient conformations. The confined space of enzymes requires FAD set in its specific intermediate conformation. However, conventional methods only detect stable conformations of FAD molecules, while transient intermediates are hidden in ensemble measurements. There still exists a challenge to uncover the transient conformation of each FAD molecule, which hinders the understanding of the structure-activity relationship of the FAD mechanism. Here, we employ the electrochemically confined space of an aerolysin nanopore to directly characterize a series of transient conformations of every individual FAD. Based on distinguishable current blockages, the "stack", "open", and four quasi-stacked FADs are clearly determined in solution, which is further confirmed by temperature-dependent experiments and mutant aerolysin assay. Combined with molecular dynamics simulations, we achieved a direct correlation between the residual current ratio (/ ) and FAD backbone angle. These results would facilitate further understanding of the structure-activity relationship in the flavoprotein.
黄素腺嘌呤二核苷酸（FAD）作为一种辅因子，参与众多重要的代谢途径，其生物学功能与其瞬时构象内在相关。酶的受限空间要求FAD处于其特定的中间构象。然而，传统方法只能检测FAD分子的稳定构象，而瞬时中间体则隐藏在整体测量中。揭示每个FAD分子的瞬时构象仍然存在挑战，这阻碍了对FAD机制的构效关系的理解。在此，我们利用气溶素纳米孔的电化学受限空间直接表征每个单独FAD的一系列瞬时构象。基于可区分的电流阻断，溶液中清晰地确定了“堆叠”、“开放”和四种准堆叠的FAD，这通过温度依赖性实验和突变气溶素测定得到进一步证实。结合分子动力学模拟，我们实现了残余电流比（/）与FAD主链角度之间的直接关联。这些结果将有助于进一步理解黄素蛋白中的构效关系。
