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Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations

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Abstract

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK A values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain 13Cγ nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK A values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK Ai values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK A values and hence catalytic roles of these two residues result from their electrostatic coupling.

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Acknowledgments

We are grateful Lewis Kay for friendship, expert advice, and countless pulse sequences, without which none of our work would be possible. Yom Huledet Same’ach! We also thank Rick Dahlquist and Steve Withers for insightful comments and Philip Johnson for early experimental help. This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC; to LPM). 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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Authors and Affiliations

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

    Lawrence P. McIntosh, Daigo Naito, Simon J. Baturin, Mark Okon & Manish D. Joshi

  2. School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

    Jens E. Nielsen

Authors
  1. Lawrence P. McIntosh
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  2. Daigo Naito
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  3. Simon J. Baturin
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  4. Mark Okon
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  5. Manish D. Joshi
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  6. Jens E. Nielsen
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Correspondence to Lawrence P. McIntosh.

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McIntosh, L.P., Naito, D., Baturin, S.J. et al. Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations. J Biomol NMR 51, 5 (2011). https://doi.org/10.1007/s10858-011-9537-x

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