The interaction between IgE and its high affinity receptor (Fc⑀RI) is a critical step in the development of allergic responses. Detailed characterization of the IgEFc⑀RI interaction may offer insights into possible modes of inhibiting the interaction, which could thereby act as a potential therapy for allergy. In this study, NMR, CD, and fluorescence spectroscopies have been used to characterize structurally the C⑀3 domain of IgE and its interaction with other protein ligands, namely, C⑀2, C⑀4, sFc⑀RI␣, and CD23. We have shown that the recombinant C⑀3 domain exists alone in solution as a "molten globule." On interaction with sFc⑀RI␣, C⑀3 adopts a folded tertiary structure, as shown by the release of the fluorescent probe 8-anilinonaphthalene-1-sulfonate and by characteristic changes in the 1 H, 15 N heteronuclear single quantum coherence NMR spectrum. However, the interactions between the C⑀3 domain and C⑀2, C⑀4, or CD23 do not induce such folding and would therefore be expected to involve only local interaction surfaces. The conformational flexibility of the C⑀3 domain of the whole IgE molecule may play a role in allowing fine tuning of the affinity and specificity of IgE for a variety of different physiological ligands and may be involved in the conformational change of IgE postulated to occur on interaction with Fc⑀RI.
The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm::HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques.
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