Urea-driven conformational changes in surface-bound superoxide dismutase

Both surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM) have been used to observe the change in Cu/Zn superoxide dismutase (SOD1) tethered to a Au film upon urea-induced denaturation. Exposure to a urea solution causes denaturation of SOD1, which shifts the minimum in the SPR curve to a larger angle without any change in reflectivity at the resonant angle (θ_SPR) for different urea concentrations. The differential reflectivity at θ_SPR (Δ(R _min/R_o)) increases sigmoidally as a function of urea concentration becoming saturated at concentrations above 4 M. With the assumption of a two-state model for the denaturation of SOD1, the Gibbs free energy change for the denaturation of SOD1 on the Au surface is estimated to be ΔG^o = 1.8 ± 0.7 kcal/mol, which is lower by approximately one order of magnitude than that of SOD1 in the bulk solution. The immobilized SOD1 on the Au surface can be reversibly denatured and renatured. Consistent with calculations based on Fresnel equations for a multilayer system, liquid-AFM images show that upon denaturation, the thickness of the tethered SOD1 increases by ca. 2.0 times. Thus, SOD1 on the Au film tries to stretch its polypeptide chain in the vertical direction on unfolding.