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

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⁺ (q_tot = 0.7) than Cs⁺ (q_tot = 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.