Abstract
C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca(2+)-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mM Ca(2+) exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s(20,w)(0) of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca(2+), CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R(G) values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K(D) value of 21 microM in solution (140 mM NaCl, 2 mM Ca(2+)) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K(D) value of 23 microM (140 mM NaCl, 2 mM Ca(2+)), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mM Ca(2+) and 140 mM NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.
Highlights
Interacts with immunoglobulin receptors to recruit phagocytic cells, in addition to exerting regulatory effects on neutrophils [5]
C-reactive protein (CRP) Pentamer-Decamer Equilibrium plinary strategy based on analytical ultracentrifugation (AUC) and x-ray scattering in solution, and surface plasmon resonance (SPR) on surfaces established the self-association state of CRP in a concentration range of 0.1–5.4 mg/ml in 2 mM Ca2ϩ
Using previously established methods [29, 30], we show from AUC size distribution analyses c(s) that pentamers and decamers of CRP are readily identified, whereas constrained x-ray scattering modeling quantified the amounts of pentamers and decamers in solution
Summary
Purification of CRP—Human CRP was isolated and purified as described previously [32]. Samples were extensively dialyzed into 10 mM Tris buffers (with or without 2 mM CaCl2) containing 50 mM NaCl, 140 mM NaCl, or 250 mM NaCl, all at pH 8.0, for AUC and x-ray scattering data acquisition. Sedimentation coefficients were calculated from the x-ray hydrated sphere models using the HYDRO program [42, 45], and from the crystal coordinates using HYDROPRO software [46]. Native CRP stored in 140 mM NaCl buffer containing 2 mM Ca2ϩ was coupled to the flow cell of a carboxylated dextran (CM5) research grade sensor chip via a standard amine coupling procedure according to the manufacturer’s protocol. Binding and equilibrium analyses using these CRP-immobilized chips were performed at 25 °C in duplicate or triplicate runs using the appropriate Biacore X100 wizards at flow rates of 10 –30 l/min. Running buffer was either Tris-buffered saline (10 mM Tris, 140 mM NaCl, 0.005% P20 surfactant, pH 8.0) or HBS (HEPES-buffered saline, 10 mM HEPES, 50 mM NaCl, 0.005% P20 surfactant, pH 8.0), with or without 2 mM CaCl2
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