Monitoring conformational changes of immobilized RNase A and lysozyme in reductive unfolding by surface plasmon resonance.
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.
Chen
Liang-Yu
LY
Department of Biotechnology, Ming-Chuan University, No. 5, Teh-Ming Road, Gui-Shan 333, Taiwan. loknath@mail.mcu.edu.tw
D000818:Animals; D002417:Cattle; D002645:Chickens; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D004800:Enzymes, Immobilized; D012259:Ribonuclease, Pancreatic; C421864:hen egg lysozyme; D009113:Muramidase