Monitoring conformational changes of immobilized RNase A and lysozyme in reductive unfolding by surface plasmon...

A labeling-free surface plasmon resonance (SPR) sensor technique was used to monitor the conformational changes of immobilized globular proteins (RNase A and Lysozyme) in chemical unfolding and refolding. The conformational changes of proteins at solid/liquid interface are characterized as two-state transformation (S-shaped) curves through matrix-effect correction and theoretic estimation. By extrapolation with a Santoro-Bolen equation, the SPR results for both reductive immobilized proteins are estimated to 1.9 kcal mole(-1) global free energy (DeltaG(U)) in urea-induced unfolding. But the DeltaG(U) for RNase A and Lysozyme in GdmCl-induced unfolding are 1.5 and 2.15 kcal mole(-1), respectively. The disagreement in free energy is partially accounted for by the differences of intra-molecular interactions and immobilization.