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

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


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.

• Content Type Journal Article
• Category Article
• Pages 5-19
• DOI 10.1007/s10858-011-9537-x
• Authors
  • Lawrence P. McIntosh, 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
  • Daigo Naito, 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
  • Simon J. Baturin, 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
  • Mark Okon, 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
  • Manish D. Joshi, 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
  • Jens E. Nielsen, School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738
  • Journal Volume Volume 51
  • Journal Issue Volume 51, Numbers 1-2

Source: Journal of Biomolecular NMR