1 research outputs found
Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy
Plant type [2Fe-2S] ferredoxins function
primarily as electron
transfer proteins in photosynthesis. Studying the unfolding–folding
of ferredoxins in vitro is challenging, because the unfolding of ferredoxin
is often irreversible due to the loss or disintegration of the iron–sulfur
cluster. Additionally, the in vivo folding of holo-ferredoxin requires
ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based
single-molecule force microscopy and protein engineering techniques
to directly study the mechanical unfolding and refolding of a plant
type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in
a three-state mechanism. The first step is the unfolding of the protein
sequence that is outside and not sequestered by the [2Fe-2S] center,
and the second one relates to the force-induced rupture of the [2Fe-2S]
metal center and subsequent unraveling of the protein structure shielded
by the [2Fe-2S] center. During repeated stretching and relaxation
of a single polyprotein, we observed that the completely unfolded
ferredoxin can refold to its native holo-form with a fully reconstituted
[2Fe-2S] center. These results demonstrate that the unfolding–refolding
of individual ferredoxin is reversible at the single-molecule level,
enabling new avenues of studying both folding–unfolding mechanisms,
as well as the reactivity of the metal center of metalloproteins in
vitro