Metal-Ion-Specific Screening of Charge Effects in Protein Amide H/D Exchange and the Hofmeister Series

In this study, protein charge ladders and mass spectrometry were used to quantify how metal cations in the Hofmeister series (Na⁺, K⁺, Li⁺, Mg²⁺, and Ca²⁺) permute the effects of lysine acetylation on the rate of amide H/D exchange in a representative protein (myoglobin, Mb). The successive acetylation of up to 18 Lys-ε-NH₃⁺ groups in Mb caused a linear decrease in its global rate of amide H/D exchange (as measured by mass spectrometry), despite also decreasing the thermostability of Mb by >10 °C. The ability of a metal cation to screen kinetic electrostatic effects during H/D exchangeand to abolish the protective effect of acetylation against H/D exchangewas found to depend on the position of the cation in the Hofmeister series. Na⁺ and K⁺ cations did not fully equalize the rates of H/D exchange among each “rung” of the charge ladder, whereas Mg²⁺ and Ca²⁺ did equalize rates without eliminating the hydrophobic core of the protein (i.e., without unfolding Mb); Li⁺ exhibited intermediate effects. The ability of Mg²⁺ and Ca²⁺ to completely screen electrostatic effects associated with the H/D exchange of charge isomers of Mb suggests that Mg²⁺ or Ca²⁺ (but not Na⁺ or K⁺) can be used to quantify the magnitude by which electrostatic charge contributes to the observed rates of amide H/D exchange in proteins.