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The binding affinity between each of the engineered IpaD alanine mutants and the stable IpaB28-226 construct was measured using fluorescence polarization. Here, the DOC effect on binding affinity between IpaB and the engineered IpaD π-helix mutants was quantified by holding IpaB28-226-Alexa568 concentration constant while the concentration of IpaD or engineered IpaD mutant was titrated from 0-10 μM with identical conditions then tested in the presence of 1 mM DOC.
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