Insensitivity of Perturbed Carboxyl pK_a Values in the Ovomucoid Third Domain to Charge Replacement at a Neighboring Residue

Abstract

A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pK(a) values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pK(a) values. However, the expected elevation in pK(a) values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pK(a) values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4-0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5-11 A away from Lys 34. Two-dimensional (1)H NMR techniques were used to determine pK(a) values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pK(a) values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pK(a) values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.

Accurate, conformation-dependent predictions of solvent effects on protein ionization constants

“…The insensitivity of the pKas of three acidic residues (Asp-7, Glu-10, Glu-19) to the removal of a neighboring positively charged residue (Lys 34) was not accurately predicted by simple FDPB calculations (20). FDPB MF predicted smaller pKa perturbations (Table 4), with rmsd and maximal error from the experimental data of 0.28 and 0.4 pH unit, respectively.…”

Carboxyl pKa Values and Acid Denaturation of BBL

“…Conventionally, pK a values are determined by monitoring the pH dependence of proton chemical shifts in 1 H-C β or 1 H-C γ correlations of aspartate or glutamate residues, respectively. [18][19][20][21] Similarly, pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1 H-13 C heteronuclear two-dimensional (2D) NMR can be used for measuring the carboxyl titration curve. 20,22,23 In general, 13 C resonances yield more accurate pK a values of carboxylic residues, 20 because 13 C chemical shifts are less sensitive than those of 1 H shifts to factors other than the changes in ionization state.…”

Role of the Charge–Charge Interactions in Defining Stability and Halophilicity of the CspB Proteins

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Moreover, several computational models were shown to be capable of reliably predicting the effects of amino acid substitutions on the protein stability. 2,4,[6][7][8][9][10]12,16,21,22 In most cases these computational models are largely based on native state structure and ignore or oversimplify the unfolded state contribution.…”