pK_a Values and the pH Dependent Stability of the N-Terminal Domain of L9 as Probes of Electrostatic Interactions in the Denatured State. Differentiation between Local and Nonlocal Interactions

Abstract

pKa values were measured for the 6 carboxylates in the N-terminal domain of L9 (NTL9) by following NMR chemical shifts as a function of pH. The contribution of each carboxylate to the pH dependent stability of NTL9 was estimated by comparing the pKa values for the native and denatured state of the protein. A set of peptides with sequences derived from NTL9 were used to model the denatured state. In the protein fragments, the pKa values measured for the aspartates varied between 3.8 and 4.1 and the pKa values measured for the glutamates varied between 4.1 and 4.6. These results indicate that the local sequence can significantly influence pKa values in the denatured state and highlight the difficulties in using standard pKa values derived from small compounds. Calculations based on the measured pKa values suggest that the free energy of unfolding of NTL9 should decrease by 4.4 kcal mol-1 when the pH is lowered from 6 to 2. In contrast, urea and thermal denaturation experiments indicate that the stability of the protein decreases by only 2.6 kcal mol-1 when the carboxylates are protonated. This discrepancy indicates that the protein fragments are not a complete representation of the denatured state and that nonlocal sequence effects perturb the pKa's in the denatured state. Increasing the salt concentration from 100 to 750 mM NaCl removes the discrepancy between the stabilities derived from denaturation experiments and the stability changes calculated from the pKa values. At high concentrations of salt there is also less variation of the pKa values measured in the protein fragments. Our results argue that in the denatured state of NTL9 there are electrostatic interactions between groups both local and nonlocal in primary sequence.

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

“…They also offer a relatively simple method for probing electrostatic interactions in the DSE [1,6,17,[23][24][25][26][27]. The approach exploits the linkage relationship that connects pH dependent changes in ΔG o with the difference in the number of protons bound to the native state and to the DSE [23]: (1) ΔQ in equation 1 is equal to the difference in the number of protons bound to the native state (Q N ) and to the DSE (Q D ) and is a function of pH.…”

“…NTL9 is a basic protein but analysis of the pH dependent stability data rules out diminished electrostatic repulsion in the native state as an explanation for the increase in stability. As noted above, mutation that abolishes the DSE interactions will make the deviation between the calculated and experimental pH stability profile much smaller [17]. This leads to an experimentally testable prediction; namely that the K12M mutation should lead to better agreement between the calculated and experimental ΔΔG 0 curves.…”

“…In most of the cases examined there are clear deviations between the observed pH dependence of ΔG o and the behavior predicted based upon a fully unfolded DSE (Table-1). Examples include CI2, barnase, Staphylococcal Nuclease (SNase), the turkey ovomucoid third domain, the N-terminal domain of L9 and a mutant of the B1 domain of protein G [1,17,[24][25][26]28]. Most of these proteins lack disulfides thus the DSE interactions are not simply a consequence of the presence of covalent cross links.…”

“…However, indirect methods normally need to be used to study the DSE under native conditions [13]. A wide range of approaches have been used including, but not limited to, the study of peptide fragments, the analysis of destabilizing point mutants or truncation mutants, amide 1 H/ 2 H exchange experiments of the native state, changes in m-values upon mutation and the analysis of the pH dependence of protein stability [14][15][16][17]. Numerous investigations have provided compelling evidence for compact DSEs with significant amounts of residual structure under native conditions.…”

Site-specific contributions to the pH dependence of protein stability

“…Such neglect of interactions between individual ionizable or polarizable groups has proved to be useful in studies involving chemical denaturants, which are known to have a profound effect on pK a values due to a reduction of the amount of residual structure. More recently, however, general shifts of Asp and Glu side-chain pK a values in unfolded states to lower than standard pK a values (by 0.3Ϫ0.4 pH units on average) have been predicted, pointing toward the presence of stabilizing electrostatic interactions within unfolded states of proteins under nondenaturing conditions (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).…”