Journal of Biomolecular NMR

, Volume 60, Issue 2–3, pp 109–129

pH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pKa measurements


DOI: 10.1007/s10858-014-9862-y

Cite this article as:
Platzer, G., Okon, M. & McIntosh, L.P. J Biomol NMR (2014) 60: 109. doi:10.1007/s10858-014-9862-y


The pKa values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the 1H, 13C, and 15N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pKa measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain 15N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.


Protein electrostatics pH titration Chemical shift Scalar coupling Deuterium isotope shift Hydrogen exchange 

Supplementary material

10858_2014_9862_MOESM1_ESM.pdf (175 kb)
Supplementary material Two supplemental tables summarizing the complete pH-dependent chemical shifts of the blocked tripeptides (Table S1) and 13C6/15N4-l-arginine (Table S2) can be found in the online version. (PDF 174 kb)

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Gerald Platzer
    • 1
    • 2
    • 3
    • 4
  • Mark Okon
    • 1
    • 2
    • 3
  • Lawrence P. McIntosh
    • 1
    • 2
    • 3
  1. 1.Department of Biochemistry and Molecular Biology, Life Sciences Centre, 2350 Health Sciences MallUniversity of British ColumbiaVancouverCanada
  2. 2.Department of ChemistryUniversity of British ColumbiaVancouverCanada
  3. 3.Michael Smith LaboratoriesUniversity of British ColumbiaVancouverCanada
  4. 4.Department of Structural and Computational Biology, Max F. Perutz LaboratoriesUniversity of ViennaViennaAustria

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