High tolerance for ionizable residues in the hydrophobic interior of proteins

Abstract

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK(a) values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.

Fig. 1.

Thermodynamic linkage between pH dependence of stability and shifts in pK_a values. (A) Simulated pH dependence of stability (ΔG°_H2O) of a protein (thick line) and of one of its variants with an internal Lys with a pK_a value of 6 (thin line). (B) Difference in the thermodynamic stability curves (ΔΔG°_H2O) in A. (C) H⁺ titration curves (thin lines) of a Lys with a normal pK_a value of 10.4 and with a pK_a value of 6.0. The area between these two titration curves (dark line) is equivalent to ΔΔG°_H2O in B.

Fig. 2.

Effects of internal ionizable groups on thermodynamic stability. (A–F) Thermodynamic stability (ΔG°_H2O) of variants of SNase (light gray) or RNaseH (dark gray) with internal Lys at pH 10 (A), internal Glu at pH 5 (B), internal Asp at pH 5 (C), internal Lys at pH 7 (D), internal Glu at pH 7 (E), and internal Asp at pH 7 (F). (G and H) Ribbon diagram of SNase (G) (32) and RNaseH (H) (33) showing the internal positions that were substituted. All data were measured at 298 K in 100 mM KCl.

Fig. 3.

pH dependence of difference thermodynamic stability [ΔΔG°_H2O = ΔG°_H2O (variant) − ΔG°_H2O (background)], defined as (ΔΔΔG°_H2O/ΔpH) = (ΔΔG°_H2O, pH₁ − ΔΔG°_H2O, pH₂)/(∣pH₁ − pH₂∣). pH₁ = 7 and pH₂ = 5 for Asp and Glu or 10 for Lys. Blue circles identify positions where ΔΔΔG°_H2O/ΔpH = 0, indicating that the pK_a values are probably normal; orange circles identify positions where ΔΔΔG°_H2O/ΔpH > 0.2, indicating that the pK_a values are shifted in the direction that favors the neutral state (depressed for basic groups, elevated for acidic ones). Data are for variants with internal Lys (A), Glu (B), and Asp (C).

Fig. 4.

Far-UV CD spectra of SNase variants with internal Glu (A) and internal Lys (B). Also shown are the background protein in water (●), wild-type SNase unfolded in 6 M GdmCl (▲), and an unfolded T62P variant in water (△). The lines are meant only to guide the eye. All measurements at pH 7 in 25 mM Hepes and 100 mM KCl.