Positioning the Intracellular Salt Potassium Glutamate in the Hofmeister Series by Chemical Unfolding Studies of NTL9.

Abstract

In vitro, replacing KCl with potassium glutamate (KGlu), the Escherichia coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu- in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to the effects of other stabilizers (KCl, KF, and K2SO4) and destabilizers (GuHCl and GuHSCN). GuHSCN titrations at 20 ° C, performed as a function of the concentration of KGlu or another salt and monitored by NTL9 fluorescence, are analyzed to obtain R-values quantifying the Hofmeister salt concentration (m3) dependence of the unfolding equilibrium constant K(obs) [r-value = −d ln K(obs)/dm3 = (1/RT) dΔG(obs) ° /dm3 = m-value/RT]. r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 ° C, we determine a KGlu r-value of 3.22 m(−1) and K2SO4, KF, KCl, GuHCl, and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38, and −3.00 m(−1), respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 ° C.