Spinning Top-like Nanoprobes for Direct Visualization of Cooperative Ca2+-Binding-Induced Conformational Switching in Single Calmodulin Molecules

Understanding the rapid, domain-specific conformational dynamics of single calmodulin (CaM) molecules remains a major challenge due to the limited temporal resolution of existing single-molecule techniques. Here, we present a mechano-responsive strategy using high-speed nanoscale spinning tops (NSTs) as nanoprobes to directly resolve these dynamics. By constructing a single-molecule CaM protrusion on a protein corona-coated gold nanorod, we enabled stochastic thermally driven rotation, whose speed is sensitively modulated by conformational changes in CaM. Distinct hydrophilic and hydrophobic states of apo-CaM and Ca2+-bound CaM, respectively, give rise to characteristic rotational signatures, allowing millisecond-resolved detection of conformational switching. Kinetic analysis across single CaM molecules reveals the cooperative binding of two Ca2+ ions to the C-terminal domain, supported by a Hill coefficient of 1.81 and a binding stoichiometry of 2.43. This platform provides an approach for quantifying protein-ligand interactions and conformational kinetics at the single-molecule level, offering new insights into calcium-mediated signaling and dynamic protein function.