pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability

Abstract

Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using (1)H-(13)C heteronuclear NMR in a structural context. The pK(a) values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK(a) values can explain the pH dependence of protein stability but require pK(a) shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK(a) value. We show also that (13)C backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.

FIGURE 1

Titration curve of E19 analyzed with a two-state model, Eq. 2 (solid curve). The pK_a values for the shift change at high pH was determined as 8.9 in low salt (•) and to 9.0 in 0.5 M salt (○).

FIGURE 2

Structure of PGB1-QDD with all charged side-chain groups explicitly shown prepared from the Protein Data Bank file 1PGB (42). Carboxyl groups are black and lysine residues are red. pK_a differences compared to model values are indicated in the backbone where red indicates upshifted and blue downshifted. The mutated residues were modeled into the structure using Swiss model (43) and the figure was prepared using MOLMOL software (44).

FIGURE 3

Protein net charge of folded PGB1-QDD according to model pK_a values (solid curve), pK_a values in low salt (solid curve with solid circles), and pK_a values in high salt (solid curve with open circles). Protein net charge of wt PGB1 according to model pK_a values (dashed curve) and pK_a values from (5) (solid curve with triangles). (Inset) pI for the different proteins and models.

FIGURE 4

(a) ¹³C backbone carbonyl chemical shifts A48 (○) and D47 (▴) with ¹³C side-chain carboxyl chemical shift of D47 (•). (b) The hydrogen bond between K10 and E56 (•) is displayed in pH-dependent ¹³C backbone carbonyl chemical shift of K10 (▴). In a and b, Eq. 1 was fit to data and is shown as a solid curve.

FIGURE 5

Generated stability curves for PGB1-QDD based on experimental pK_a values for the native state and calculated pK_a values for the unfolded state (Table 1), which are compared to pH-dependent denaturation data (7). (a) Curves generated in low salt where the unfolded state is modeled as a Gaussian chain (dashed), model pK_a values (dashed-dotted) Gaussian chain but the pK_a value of D37 is shifted to 6.0 (solid), and a uniform pK_a shift of 0.2 units (dotted). All curves in a are generated using a pK_a value of 8.9 for the N-terminus in the folded state. (b) Curves generated in 0.5 M salt where the pK_a values of the unfolded state were modeled as a Gaussian chain but where the pK_a value of the N-terminus is 9.0 (solid) and 7.5 (dashed). Experimental stability data in low salt (•) and 2 M NaCl (▴). In a, the calculated curves were shifted to minimize the rms deviation between experimental and calculated data points. In b, the curve was shifted to fit experimental data at pH >5.