Chemistry on a Single Protein, Vascular Cell Adhesion Molecule-1, during Forced Unfolding*

Proteins of many types experience tensile forces in their normal function, and vascular cell adhesion molecule-1 (VCAM-1) is typical in this. VCAM has seven Ig domains, and each has a disulfide bond (-S–S-) buried in its core that covalently stabilizes about half of each domain against unfolding. VCAM is extended here by single molecule atomic force microscopy in the presence or absence of reducing agents. In the absence of reducing agent, a sawtooth pattern of forced unfolding reveals an average period and total length consistent with disulfide locations in VCAM. With increasing reducing agent, accessible disulfides are specifically reduced (to SH); the average period for unfolding increases up to saturation together with additional metrics of unfolding. Steered molecular dynamics simulations of unfolding indeed show that the core disulfide bond is solvent-exposed in the very earliest stages of protein extension. Michaelis-Menten kinetics emerge with reduction catalyzed by force (τ_reduction ∼ 10^–4 s). The results establish single molecule reduction, one bond at a time, and show that mechanical forces can play a key role in modulating the redox state of cell adhesion proteins that are invariably stressed in cell adhesion.