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Investigating the folding-unfolding mechanisms of metalloproteins with single-molecule force spectroscopy Li, Jiayu


Metalloproteins account for more than one-third of all proteins in nature and play important roles in biological processes. The folding process of metalloproteins is complicated, as it is driven by not only the polypeptide chain folding effect but also the metal coordination. Folding into the native structures with correctly assembled metal cofactors is a prerequisite for metalloproteins to perform their biological functions. Therefore, understanding the folding-unfolding mechanisms of metalloproteins is of critical importance. Over the past two decades, single-molecule force spectroscopy (SMFS) has evolved into a powerful method to investigate the folding-unfolding mechanisms of metalloproteins at the single-molecule level. This thesis presents the SMFS studies on the folding-unfolding mechanisms of four important metalloproteins, including three iron-sulfur proteins and one heme-containing protein. First, we studied the mechanical unfolding behavior of a high potential iron-sulfur protein by atomic force microscopy (AFM)-based SMFS, and revealed a detailed mechanical unfolding mechanism. In combination with previous studies, we proposed a general mechanical unfolding mechanism for the iron-sulfur protein family. We then investigated the folding behavior of the simplest iron-sulfur protein, rubredoxin, with optical tweezers (OT)-based SMFS. We discovered a novel binding-folding-reconstitution mechanism of the folding of rubredoxin, and highlighted the critical importance of the two-coordinate ferric site in the folding of rubredoxin. We also explored the folding behavior of another iron-sulfur protein, ferredoxin, with OT-based SMFS. The unfolded ferredoxin was found to mostly misfold instead of folding back to its native structure; however, the successful reconstitution of the β-sheet or the coordination center was observed in rare cases. In addition, we studied the folding-unfolding behavior of a heme-containing protein, cytochrome c, using OT-based SMFS. We revealed a detailed folding-unfolding mechanism for holo-form cytochrome c, and identified the deviation from random coil behavior of apo-form cytochrome c, which was previously inaccessible by ensemble spectroscopic studies. Finally, conclusions and future directions on investigating the folding-unfolding mechanisms of metalloproteins with SMFS were presented. Overall, this thesis advances our understanding of the folding-unfolding mechanisms of iron-sulfur proteins, heme-containing proteins, as well as metalloproteins in general, and the systematic studies pave the way for further research in this important area.

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