TY - JOUR
T1 - Remeasuring HEWL pKa values by NMR spectroscopy
T2 - methods, analysis, accuracy, and implications for theoretical pKa calculations
AU - Webb, Helen
AU - Tynan-Connolly, Barbara Mary
AU - Lee, Gregory M
AU - Farrell, Damien
AU - O'Meara, Fergal
AU - Søndergaard, Chresten R
AU - Teilum, Kaare
AU - Hewage, Chandralal
AU - McIntosh, Lawrence P
AU - Nielsen, Jens Erik
N1 - Copyright © 2010 Wiley-Liss, Inc.
PY - 2011/3
Y1 - 2011/3
N2 - 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.
AB - 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.
KW - Algorithms
KW - Nuclear Magnetic Resonance, Biomolecular
KW - Protein Conformation
KW - Reproducibility of Results
U2 - 10.1002/prot.22886
DO - 10.1002/prot.22886
M3 - Journal article
C2 - 21287606
SN - 0887-3585
VL - 79
SP - 685
EP - 702
JO - Proteins: Structure, Function, and Bioinformatics
JF - Proteins: Structure, Function, and Bioinformatics
IS - 3
ER -