title: A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system.
abstract: 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 analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing alpha-helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular beta-sheets at pH 2.0. Our experiments indicate that EspA has the potential to spontaneously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Kato
authors.forename: Daizo
authors.initials: D
authors.lastname: Hamada
authors.forename: Takashi
authors.initials: T
authors.lastname: Fukui
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayashi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Honda
authors.forename: Yoshikatsu
authors.initials: Y
authors.lastname: Murooka
authors.forename: Itaru
authors.initials: I
authors.lastname: Yanagihara
mesh_terms: D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D019453:Escherichia coli O157 / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
chemical_list: D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea