| The metal ions or metal clusters in metalloproteins can form a variety of metal ligand bonds with amino acid side chains,providing unique structural functions for proteins.Learning the mechanical properties of metal ligand bonds has broad significance for understanding the folding mechanisms and functional properties of metalloproteins,and lays the theoretical foundation for the design and development of new functional proteins.In particular,heme proteins play an important role in various biological processes such as electron transfer by forming metal ligand bonds to carry iron ions.Cyt ochrome b562(Cyt b562),a natural heme protein,has become an ideal target for protein engineering to modify the functional properties of heme proteins due to its simple structure,high expression and the presence of heme group.In Cyt b562,the heme group is non-covalently bound by forming Fe-S and Fe-N iron ligand bonds with the protein.The addition of the characteristic motif-CXXCH-using fixed-point mutations can transform the protein into the c-type protein Cyt c-b562,whose heme group can form thioether bonds covalently with the mutated Cys residue in addition to forming metal ligand bonds,improving the stability of the protein structure.This project combined protein engineering and single-molecule force spectroscopy based on atomic force microscopy to reveal the mechanical unfolding mechanism of Cyt b562 protein and its variants,and investigated the mechanical properties of metal ligand bonds in them,which elucidated the regulatory role of heme binding on the structural stability of the protein at the molecular level.The results showed that the Apo-Cyt b562 protein structure exhibited low intensity mechanical stability.After binding of the heme group,the structural stability of the protein was remarkably enhanced,with preferential breakage during unfolding from the Fe-N（His102）bond at the C-terminus,which is less mechanically stable（57 pN）.The Cyt c-b562 protein significantly enhanced the stability of the protein structure due to the presence of covalent thioether bonds,which caused the protein to break from the iron ligand bond formed by the N-terminal amino acid at position 7 under a force of more than105 pN.Finally,the mechanical properties of the different iron ligand bonds formed at the amino acid residues at position 102 of the protein were found to be slightly different by comparison,with Fe-S ligand bond exhibiting a stronger mechanical stability.The present work has opened up a new way to study the structural stability of such heme proteins and laid a theoretical foundation for the design and development of new functional proteins. |
