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pH-Dependent structural changes forEscherichia coliO157:H7 EspA were characterized by CD, 8-anilino-2-naphthyl sulfonic acid (ANS) fluores-cence, and sedimentation equilibrium ultracentrifugation. Far- and near-UV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures. An increase in ANS fluorescence intensity (in the presence of EspA) was observed at aci-dic pH; | ềFEBS Journal A pH-dependent conformational change in EspA a component of the Escherichia coli O157 H7 type III secretion system Tomoaki Kato1 2 Daizo Hamada2 Takashi Fukui2 Makoto Hayashi1 Takeshi Honda3 Yoshikatsu Murooka1 and Itaru Yanagihara2 1 Department of Biotechnology Graduate Schoolof Engineering Osaka University Japan 2 Department of DevelopmentalInfectious Diseases Research Institute Osaka MedicalCenter for Maternaland Child Health Japan 3 Department of BacterialInfections Research Institute for MicrobialDiseases Osaka University Japan Keywords ANS binding CD FT-IR partially unfolded sedimentation equilibrium Correspondence D. Hamada Department of Developmental Infectious Diseases Research Institute Osaka Medical Center for Maternal and Child Health 840 Murodo Izumi Osaka 594-1011 Japan Fax 81 725 57 3021 Tel 81 725 56 1220 E-mail daizo@mch.pref.osaka.jp Received 21 September 2004 revised 1 March 2005 accepted 1 April 2005 doi 10.1111 j.1742-4658.2005.04697.x pH-Dependent structural changes for Escherichia coli O157 H7 EspA were characterized by CD 8-anilino-2-naphthyl sulfonic acid ANS fluorescence and sedimentation equilibrium ultracentrifugation. Far- and near-UV CD spectra recorded between pH 2.0 and 7.0 indicate that the protein has significant amounts of secondary and tertiary structures. An increase in ANS fluorescence intensity in the presence of EspA was observed at acidic pH whereas no increased ANS fluorescence was observed at pH 7.0. These results suggest the presence of a partially unfolded state. Interestingly urea-induced unfolding transitions monitored by far-UV CD spectroscopy showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH. Although increased ANS fluorescence was observed at pH 3.0 the urea-induced unfolding curve is similar to that found at pH 7.0. This result suggests the presence at pH 3.0 of an ordered but partially unfolded state which differs from typical molten globule. The results of .
