Large shifts in pKa values of lysine residues buried inside a protein

Abstract

Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK(a) values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK(a) values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H(+) transport. The pK(a) values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK(a) values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.

Fig. 1.

Measurement of pK_a values through linkage analysis of the pH dependence of thermodynamic stability. (A) Thermodynamic stability () of reference protein (Δ+PHS nuclease) (○) and its L125K variant (●) measured by GdnHCl denaturation monitored by Trp fluorescence. The line is from a simulation and it is only meant to guide the eye. (B) Difference in thermodynamic stability of Δ+PHS and the L125K variant (variant–reference). The line describes the fit of Eq. 2 to the data. The pK_a of Lys-125 in folded () and denatured () states are indicated. (C) Thermodynamic stability of Δ+PHS nuclease (○) and of its T62K variant (●) measured by GdnHCl denaturation monitored by Trp fluorescence. The line is from a simulation and it is only meant to guide the eye. (D) Difference in thermodynamic stability of Δ+PHS and the T62K variant (variant–reference). The line describes the fit of Eq. 3 to the data. The pK_a values relevant to Lys-62 ( and ) and the phenomenological pK_a values ( and ) of other ionizable side chain(s) affected by Lys-62 are indicated.

Fig. 2.

pK_a values of Lys at 25 internal positions. (A) pK_a values. White bars identify groups that do not exhibit a detectable shift in pK_a value. Colors are only meant to separate arbitrarily small, medium, and large shifts in pK_a values (B) Distribution of internal Lys residues in the structure of Δ+PHS nuclease [PDB ID code 3BDC (35)], color-coded according to the magnitude of the shift in pK_a relative to the normal value of 10.4 for Lys in water, as represented in A.

Fig. 3.

Conformational consequences of ionization of Lys residues at 25 internal positions. (A) pH titrations of Δ+PHS nuclease (black circle) and of variants with internal Lys (red, blue, and gray circles) residues monitored by Trp-fluorescence, as described previously (12). Variants that exhibit partial (blue circles) or global (red circles) unfolding concomitant with ionization of the internal Lys are labeled. (B) pH titrations of Δ+PHS nuclease (black circle) and of variants with internal Lys (red, blue, and gray circles) residues monitored by far-UV CD at 222 nm. Variants that exhibit partial (blue circles) or global (red circles) unfolding concomitant with ionization of the internal Lys are labeled. (C) Location of Lys residues that trigger local (blue spheres) or global (red spheres) structural changes upon ionization, mapped on the structure of Δ+PHS [PDB ID code 3BDC (35)].

Fig. 4.

Comparison of pK_a shifts of Glu and Lys at 25 internal positions. (A) Difference in absolute values of shifts in pK_a of Lys and Glu residues at 25 internal positions in SNase, calculated as (|(pK_a,Glu - 4.5)| - |(pK_a,Lys - 10.4)|). This assumes values of 4.5 and 10.4 for the normal pK_a of Glu and Lys in water, respectively. Positive values identify cases where the shifts in the pK_a of a Glu residue at a given position is greater than the shift in the pK_a of a Lys residue. The color code is meant to distinguish groups with small (green) and large (blue) differences in pK_a values. (B) Distribution of the differences in the pK_a values of Lys and Glu residues mapped on the structure of Δ+PHS [PDB ID code 3BDC (35)].