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

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Abstract

The pK a 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 pK a 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.

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Acknowledgments

G. P. was supported by the Austrian Science Fund (FWF). This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to L.P.M. Instrument support was provided by the Canadian Institutes for Health Research (CIHR), the Canadian Foundation for Innovation (CFI), the British Columbia Knowledge Development Fund (BCKDF), the UBC Blusson Fund, and the Michael Smith Foundation for Health Research (MSFHR).

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  1. Gerald Platzer

    Present address: Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter Campus 5, 1030, Vienna, Austria

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  1. Department of Biochemistry and Molecular Biology, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

    Gerald Platzer, Mark Okon & Lawrence P. McIntosh

  2. Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada

    Gerald Platzer, Mark Okon & Lawrence P. McIntosh

  3. Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada

    Gerald Platzer, Mark Okon & Lawrence P. McIntosh

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10858_2014_9862_MOESM1_ESM.pdf

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)

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Platzer, G., Okon, M. & McIntosh, L.P. pH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements. J Biomol NMR 60, 109–129 (2014). https://doi.org/10.1007/s10858-014-9862-y

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