Detection of conformational changes in an immobilized protein using surface plasmon resonance.
Utilizing surface plasmon resonance (SPR), we have developed novel methodology 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 exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins. The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12-7.80 and yielded reproducible measurements. This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor. This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.
Sota
H
H
Department of Research and Development, Amersham Pharmacia Biotech K.K., Tokyo, Japan. hiroyuki.sota@jp.apbiotech.com
Hasegawa
Y
Y
Iwakura
M
M
D015374:Biosensing Techniques; D002942:Circular Dichroism; D003911:Dextrans / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D012031:Refractometry / Q000379:methods*; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics
C113155:DHFR-ASC; D003911:Dextrans; D004800:Enzymes, Immobilized; C014392:carboxymethyl dextran; D013762:Tetrahydrofolate Dehydrogenase