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Revealing the transient conformations of a single flavin adenine dinucleotide using an aerolysin nanopore

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Abstract

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 (I/I0) and FAD backbone angle. These results would facilitate further understanding of the structure–activity relationship in the flavoprotein.

Graphical abstract: Revealing the transient conformations of a single flavin adenine dinucleotide using an aerolysin nanopore

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Publication details

The article was received on 27 Jun 2019, accepted on 20 Sep 2019 and first published on 23 Sep 2019


Article type: Edge Article
DOI: 10.1039/C9SC03163D
Chem. Sci., 2019, Advance Article
  • Open access: Creative Commons BY-NC license
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    Revealing the transient conformations of a single flavin adenine dinucleotide using an aerolysin nanopore

    M. Li, Y. Wang, Y. Ying and Y. Long, Chem. Sci., 2019, Advance Article , DOI: 10.1039/C9SC03163D

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