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

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.

Structure, dynamics, and ionization equilibria of the tyrosine residues in Bacillus circulans xylanase.

We have developed NMR spectroscopic methods to investigate the tyrosines within Bacillus circulans xylanase (BcX). Four slowly exchanging buried tyrosine hydroxyl protons with chemical shifts between 7.5 and 12.

Stability of the glycerol facilitator in detergent solutions.

Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence.