Single Molecule Force Spectroscopy Studies on Metalloproteins: Opportunities and Challenges

Metalloproteins play important roles in a wide range of biological processes. Elucidating the mechanisms via which metalloproteins fold and constitute their metal centers is critical to the understanding of the functions and dynamics of metalloproteins. Owing to its superior force and length resolution, single-molecule force spectroscopy (SMFS) has evolved into a powerful tool to probe the unfolding and folding mechanisms of metalloproteins at the single level by forcing metalloproteins to unfold and then refold along a reaction coordinate defined by the applied stretching force. The folding of metalloproteins is complex and involves two interwound processes, the folding of the polypeptide chain and the constitution of the metal center. Experimental studies of the folding of metalloproteins are challenging. SMFS studies have allowed researchers to directly probe the folding and unfolding of metalloproteins at the single-molecule level and the effect of metal centers on the folding-unfolding energy landscape of metalloproteins. New mechanistic insights on the folding and unfolding of some metalloproteins have been obtained, demonstrating the power and unique advantages that SMFS techniques may offer. In this Perspective, using calcium-binding proteins and small iron-sulfur proteins as examples, I provide a concise overview of the information and insights that SMFS studies have provided to understand the folding and unfolding of metalloproteins. I also discuss the opportunities and challenges that are present in this fast-progressing area of research.