Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor.
The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K(D)=0.03 mM and a low-affinity site with K(D)=5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K(D)=5 microM at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca2+ is an efficient regulator under physiological conditions.
Department of Biochemistry and Organic Chemistry, Uppsala University, SE-75123 Uppsala, Sweden.
Tony
T
Christopeit
Thomas
T
Gossas
U Helena
UH
Danielson
D015374:Biosensing Techniques / Q000379:methods*; D002097:C-Reactive Protein / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010767:Phosphorylcholine / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000379:methods*
D010767:Phosphorylcholine; D002097:C-Reactive Protein; D002118:Calcium