Journal Article


Jens Erik Nielsen
Lawrence P. McIntosh
Chandralal Hewage
Kaare Teilum
Chresten R Søndergaard
Fergal O'Meara
Damien Farrell
Gregory M Lee
Barbara Mary Tynan-Connolly
Helen Webb



spectroscopy analysis nmr spectroscopy electric field effects protein conformation algorithms reproducibility of results nuclear magnetic resonance biomolecular

Remeasuring HEWL pK(a) values by NMR spectroscopy: methods, analysis, accuracy, and implications for theoretical pK(a) calculations. (2010)

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.
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Full list of authors on original publication

Jens Erik Nielsen, Lawrence P. McIntosh, Chandralal Hewage, Kaare Teilum, Chresten R Søndergaard, Fergal O'Meara, Damien Farrell, Gregory M Lee, Barbara Mary Tynan-Connolly, Helen Webb

Experts in our system

Jens Erik Nielsen
University College Dublin
Total Publications: 27
Damien Farrell
University College Dublin
Total Publications: 16