Arginine residues at internal positions in a protein are always charged

Abstract

Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK(a) values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK(a) values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK(a); they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments.

Fig. 1.

Structure of Δ+PHS nuclease showing internal positions that were substituted with Arg. Spheres represent the C_α of residues substituted with Arg. Colors identify variants that were unfolded by the substitution with Arg (red), variants that were folded but enzymatically inactive (yellow), and variants that were folded and enzymatically active (blue). Bold numbers identify the positions (72, 90, and 109) that were substituted with Arg in the three crystal structures that were solved. The circle identifies the approximate location of the active site. Structure shown is Δ+PHS (PDB ID code 3BDC) (42).

Fig. 2.

Far-UV CD spectra of Arg-containing variants at pH 7 (A) and pH 10 (B). Δ+PHS nuclease (solid black line), Δ+PHS nuclease in 6 M GdnHCl (dashed black line), and the unfolded SNase variant T62P in water (dashed gray line) are shown for reference. Solid gray lines correspond to the 19 Arg variants with no apparent structural rearrangement. Variants of interest are highlighted: A58R (green); V39R, Y91R, and N100R (blue); I92R (orange); and V104R (red). GdnHCl-induced unfolding of Arg-containing variants at pH 7 (C) and pH 10 (D). Points represent the normalized fluorescence measured at 296 nm; lines represent two-state linear extrapolation model fit to each curve. Color scheme is identical to A and B. The two-state model could not be fit to the titration curves of the I92R or V104R variants.

Fig. 3.

Crystal structures of the variants with I72R (A and D), A90R (B and E), and A109R (C and F) substitutions. (Top) The effect of the Arg substitution on the global backbone conformation of SNase. The Arg-containing variants are colored blue; the structure of the reference Δ+PHS protein is in gray. The Arg side chains are shown in stick models. (Bottom) The polar contacts made by the Arg side chains. Water molecules are shown as red spheres. Final 1.25σ 2F_o - F_c electron density is shown in blue.

Fig. 4.

Correlation in the difference in thermodynamic stability () between variants of Δ+PHS SNase with Lys or Arg at internal positions, measured at pH 5 for variants with Lys to ensure they are all charged, and at pH 7 for variants with Arg, where they are all charged. The thermodynamic stability of Δ+PHS SNase is independent of pH between pH 5 and 7 (21). The stability of values were calculated by subtracting from . The error bars are the estimated uncertainty of the measurement. Values for variants with Lys are from ref. .