Exploration of Metal-Ligand Coordination Bonds in Proteins by Single-molecule Force Spectroscopy

Thanks to the binding of various metal ions, metalloprotein plays an essential role in many different biological processes and represents an indispensable protein subgroup. Thus, the knowledge of the incorporated metal site and metal-ligand coordination bond in the protein is invaluable for understanding this critical bio-macromolecule and designing a new one. Over the last decade, atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) has been used to explore the metalloprotein as an emerging methodology, focusing on measuring metal-ligand bond strength. By stretching the protein molecule along its peptide backbone, AFM-SMFS can unfold the protein secondary structure and break and quantify the metalligand bond/metal cluster. Moreover, the very recent development of enzymatic, site-specific protein conjugation and immobilization methods for the SMFS system enables the highly efficient and accurate measurement of the metal-ligand bond strength in proteins. As a result, comparing the strength among different types of metal-ligand bonds in proteins becomes possible, and the trend of their strength is gradually revealed, such as the Fe(III)-ligand bond in iron-binding proteins. Thus, we envision that in the future AFM-SMFS may provide a unique insight to uncover the general principle of how protein selectively binds to metal ion.