Reversible Unfolding–Refolding of Rubredoxin: A Single‐Molecule Force Spectroscopy Study

Abstract

In metalloproteins, metal centers serve as active sites for a range of functional purposes and as important structural elements to facilitate protein folding and assembly. It is challenging to observe the reversible unfolding and refolding of metalloproteins because of a loss or decomposition of the metal center. Here, the reversible unfolding-refolding of the iron-sulfur protein rubredoxin was observed directly using single-molecule force spectroscopy. The results demonstrate that the iron can remain attached to the CXXC motif when rubredoxin is unfolded. Upon relaxation, the unfolded rubredoxin can refold into its native holo state with the reconstituted FeS 4 center. The possible loss of iron from the unfolded protein prevents rubredoxin from refolding into its native holo state. These results demonstrated that unfolding of rubredoxin is reversible, as long as the iron remains attached, and provide experimental evidence for the iron-priming mechanism for the folding of rubredoxin.

Force-Dependent Folding Kinetics of Single Molecules with Multiple Intermediates and Pathways

“…Single-molecule techniques have been used to characterize intermediates in a wide variety of molecular systems, from protein folding − and binding metal–metalloproteins − to RNA and DNA folding, − G-quadruplex DNA formation, , DNA duplexes formation with base-pair mismatches, and synthetic molecular foldamers and shuttles. , Moreover, upon misfolding, molecular intermediates have also been shown to play a role, for example, in neuronal calcium sensors …”