Weak protein–cationic co-ion interactions addressed by X-ray crystallography and mass spectrometry

Abstract

The adsorption of Rb+, Cs+, Mn2+, Co2+and Yb3+onto the positively charged hen egg-white lysozyme (HEWL) has been investigated by solving 13 X-ray structures of HEWL crystallized with their chlorides and by applying electrospray ionization mass spectrometry (ESI-MS) first to dissolved protein crystals and then to the protein in buffered salt solutions. The number of bound cations follows the order Cs+< Mn2+≃ Co2+< Yb3+at 293 K. HEWL binds less Rb+(qtot= 0.7) than Cs+(qtot= 3.9) at 100 K. Crystal flash-cooling drastically increases the binding of Cs+, but poorly affects that of Yb3+, suggesting different interactions. The addition of glycerol increases the number of bound Yb3+cations, but only slightly increases that of Rb+. HEWL titrations with the same chlorides, followed by ESI-MS analysis, show that only about 10% of HEWL binds Cs+and about 40% binds 1–2 Yb3+cations, while the highest binding reaches 60–70% for protein binding 1–3 Mn2+or Co2+cations. The binding sites identified by X-ray crystallography show that the monovalent Rb+and Cs+preferentially bind to carbonyl groups, whereas the multivalent Mn2+, Co2+and Yb3+interact with carboxylic groups. This work elucidates the basis of the effect of the Hofmeister cation series on protein solubility.