caa a22 4500 445325496 CHVBK 20180317142719.0 cr unu---uuuuu 170323e20110901xx s 000 0 eng 10.1007/s10858-011-9537-x doi (NATIONALLICENCE)springer-10.1007/s10858-011-9537-x Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations [Elektronische Daten] [Lawrence McIntosh, Daigo Naito, Simon Baturin, Mark Okon, Manish Joshi, Jens Nielsen] 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. Springer Science+Business Media B.V., 2011 Acid-base equilibria nationallicence Glycoside hydrolase nationallicence pH-dependent chemical shift nationallicence pH titration nationallicence Protein electrostatics nationallicence McIntosh Lawrence Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut Naito Daigo Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut Baturin Simon Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut Okon Mark Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut Joshi Manish Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut Nielsen Jens School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland aut Journal of Biomolecular NMR Springer Netherlands 51/1-2(2011-09-01), 5-19 0925-2738 51:1-2<5 2011 51 10858 https://doi.org/10.1007/s10858-011-9537-x text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9537-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- McIntosh Lawrence Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- Naito Daigo Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- Baturin Simon Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- Okon Mark Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- Joshi Manish Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- Nielsen Jens School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 51/1-2(2011-09-01), 5-19 0925-2738 51:1-2<5 2011 51 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer