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

TitleDissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations
Publication TypeJournal Article
Year of Publication2011
AuthorsMcIntosh, LP, Naito, D, Baturin, SJ, Okon, M, Joshi, MD, Nielsen, JE
JournalJOURNAL OF BIOMOLECULAR NMR
Volume51
Pagination5-19
Date PublishedSEP
ISSN0925-2738
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 (13)C(gamma) 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.

DOI10.1007/s10858-011-9537-x