Cooperative Motion of a Multi-Subunit Protein Visualized by X-Ray Single Molecule Tracking

Abstract

Most of proteins or enzymes are multimeric proteins, such as dimer, trimer, or higher-order structures (Goodsell et al. Annu Rev Biophys Biomol Struct, 2000). Usually, these proteins have multiple biding sites for ligands, such as ATP, peptide and others, that induce protein's conformational change. The function of the protein is regulated by such conformational changes that may give rise to cooperativity. Therefore, assessment of the cooperativity between subunits of proteins is important to understand the function of complex proteins.We have previously reported that the diffracted X-ray tracking (DXT) method could trace ATP-induced twisting motion of group II chaperonin ring at a single molecule level with high accuracy. In DXT, a gold nanocrystal immobilized on one side of chaperonin-ring is used as tracer for structural change of chaperonin. Our analyses clearly showed that the chaperonin ring partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2-6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. In this study, we checked how ATPase deficient mutant subunits modulate the speed or frequency of twisting motion. We controlled the number of ATPase deficient mutant within one chaperonin ring, constructed the ATP deficient hetero-ring using circular permutated connected mutants, and evaluated the effects of those mutants to chaperonin-ring's twisting motion. We found that the equivalent twisting motion was observed in hetero-ring chaperonin and inter-ring communication is dispensable for the function of group II chaperonin3.