DETECTION OF CONFORMATIONAL-CHANGES IN AN IMMOBILIZED PROTEIN USING SURFACE-PLASMON RESONANCE

Utilizing surface plasmon resonance (SPR), we have developed novel met hodology for the detection of conformational change(s) in immobilized proteins. A genetically altered E. coli dihydrofolate reductase (DHFR- ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus. The DHFR-ASC-immobilized surface exhi bited a larger response to acid treatment than reference surfaces lack ing immobilized proteins. The SPR signal of the tethered protein and t he molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR s ignal reflect conformational changes occurring during acid denaturatio n. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered pro tein sensor surface was stable to repeated acid treatment using soluti ons in the pH range of 0.12-7.80 and yielded reproducible measurements . This is the first demonstration of detection of conformational chang es in an immobilized protein using an SPR biosensor. This technique ha s potential for developing novel sensors and/or switching devices in r esponse to protein conformational changes.