@ARTICLE{Webb2011,
  author = {Webb, Helen and Tynan-Connolly, Barbara Mary and Lee, Gregory M and
	Farrell, Damien and O'Meara, Fergal and S\o{}ndergaard, Chresten R
	and Teilum, Kaare and Hewage, Chandralal and McIntosh, Lawrence P
	and Nielsen, Jens Erik},
  title = {{Remeasuring HEWL pK(a) values by NMR spectroscopy: methods, analysis,
	accuracy, and implications for theoretical pK(a) calculations.}},
  journal = {Proteins: Struct., Funct., Bioinf.},
  year = {2011},
  volume = {79},
  pages = {685--702},
  number = {3},
  month = mar,
  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.},
  doi = {10.1002/prot.22886},
  issn = {1097-0134},
  keywords = {Algorithms,Biomolecular,Biomolecular: methods,HEWL,Nuclear Magnetic
	Resonance,Protein Conformation,Reproducibility of Results},
  pmid = {21287606},
}

