| Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations. | |
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MedLine Citation:
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PMID: 21947911 Owner: NLM Status: In-Data-Review |
Abstract/OtherAbstract:
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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 (13)C(γ) 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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Authors:
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Lawrence P McIntosh; Daigo Naito; Simon J Baturin; Mark Okon; Manish D Joshi; Jens E Nielsen |
Publication Detail:
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Type: Journal Article Date: 2011-09-27 |
Journal Detail:
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Title: Journal of biomolecular NMR Volume: 51 ISSN: 1573-5001 ISO Abbreviation: J. Biomol. NMR Publication Date: 2011 Sep |
Date Detail:
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Created Date: 2011-09-28 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9110829 Medline TA: J Biomol NMR Country: Netherlands |
Other Details:
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Languages: eng Pagination: 5-19 Citation Subset: IM |
Affiliation:
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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, mcintosh@chem.ubc.ca. |
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