C-reactive protein exists in an NaCl concentration-dependent pentamer-decamer equilibrium in physiological buffer

Abstract

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca2+-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 Ca2+ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s20,w0 of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca2+, 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 RG values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged KD value of 21 μM in solution (140 mM NaCl, 2 mM Ca2+) 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 KD value of 23 μM (140 mM NaCl, 2 mM Ca2+), 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 Ca2+ 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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.