Remeasuring HEWL pK_a values by NMR spectroscopy: Methods, analysis, accuracy, and implications for theoretical pK_a calculations

Abstract

Site-specific pK(a) values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH-dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pK(a) calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nuclei versus sample pH. However, careful analysis of these curves is needed to extract residue-specific pK(a) values since pH-dependent chemical shift changes can arise from many sources, including through-bond inductive effects, through-space electric field effects, and conformational changes. We have re-measured titration curves for all carboxylates and His 15 in Hen Egg White Lysozyme (HEWL) by recording the pH-dependent chemical shifts of all backbone amide nitrogens and protons, Asp/Glu side chain protons and carboxyl carbons, and imidazole protonated carbons and protons in this protein. We extracted pK(a) values from the resulting titration curves using standard fitting methods, and compared these values to each other, and with those measured previously by ¹H NMR (Bartik et al., Biophys J 1994;66:1180–1184). This analysis gives insights into the true accuracy associated with experimentally measured pK(a) values. We find that apparent pK(a) values frequently differ by 0.5–1.0 units depending upon the nuclei monitored, and that larger differences occasionally can be observed. The variation in measured pK(a) values, which reflects the difficulty in fitting and assigning pH-dependent chemical shifts to specific ionization equilibria, has significant implications for the experimental procedures used for measuring protein pK(a) values, for the benchmarking of protein pK(a) calculation algorithms, and for the understanding of protein electrostatics in general.

Constant pH Replica Exchange Molecular Dynamics in Explicit Solvent Using Discrete Protonation States: Implementation, Testing, and Validation

“…The agreement is significantly better than the predictions from the implicit solvent calculations over a similar time scale. With the exception of aspartate 119 (and aspartate 52 in the 150 ns simulation), the predicted p K a values of all residues were within 1 p K unit from the experimental values given in ref (49). …”

“…Because the protonation state sampling is performed with the same GB potential in both the original and proposed methods, any differences in the predicted p K a values must be caused by differences in conformational sampling. Given the rigorous nature of the experimental measurements, 49 this provides strong evidence that the proposed method improves significantly upon the original by improving conformational sampling with an explicit solvent representation.…”

Multiscale Modeling of a Conditionally Disordered pH-Sensing Chaperone

“…pK a values from pH-REX of the unfolded monomer models do not significantly deviate from the model compound values (4.0 for Asp and 4.4 for Glu 19 ) and are somewhat closer than the dimer pK a values to NMR measurements (Table SI). The AUE of the dimer and monomer pK a values from pH-REX when compared to the NMR values all fall within the estimated uncertainty of 0.5 pK a units from measuring pK a values by various NMR methods 20 . This observation implies that just a handful of acidic residues exhibit anomalous protonation behavior and thus act as key “pH triggers” during HdeA chaperone activation.…”

Protein Dielectric Constants Determined from NMR Chemical Shift Perturbations

“…To extract Δδ tot values that originate from the titration of a single ionizable group, pH-dependent chemical shifts reported by a 15 N or 1 H N nucleus were fitted using an automated F-test based algorithm 45,46 . Only Δδ tot values with a magnitude above the uncertainty level for the specific nucleus (0.1 ppm for 15 N and 0.03 ppm for 1 H N (ref.…”