Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations.
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Date
2011Author
McIntosh, LP
Naito, D
Baturin, SJ
Okon, M
Joshi, MD
Nielsen, JE
Type
ArticleLanguage
enMetadata
Show full item recordAbstract
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.
URI
http://www.ncbi.nlm.nih.gov/pubmed/21947911http://erepository.uonbi.ac.ke:8080/xmlui/handle/123456789/52662
Citation
J Biomol NMR. 2011 Sep;51(1-2):5-19. doi: 10.1007/s10858-011-9537-x. Epub 2011 Sep 27Publisher
University of Nairobi Faculty of medicine
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- Faculty of Health Sciences (FHS) [10378]