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Biophysical Characterization of the Enzyme I of the Streptomyces coelicolor Phosphoenolpyruvate:Sugar Phosphotransferase System

Estefanía Hurtado-Gómez ^*, Gregorio Fernández-Ballester ^*, Harald Nothaft , Javier Gómez ^*, Fritz Titgemeyer  and José Luis Neira ^* 

^* Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain; Lehrstuhl für Mikrobiologies, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; and Biocomputation and Complex Systems Physics Institute, Zaragoza, Spain

Correspondence: Address reprints requests to José L. Neira, Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, Avda. del Ferrocarril s/n, 03202, Elche (Alicante), Spain. Tel.: 34-966658459; Fax: 34-966658758; E-mail: jlneira{at}umh.es.

The first protein in the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system is the homodimeric 60-kDa enzyme I (EI), which autophosphorylates in the presence of PEP and Mg²⁺. The conformational stability and structure of the EI from Streptomyces coelicolor, EI^sc, were explored in the absence and in the presence of its effectors by using several biophysical probes (namely, fluorescence, far-ultraviolet circular dichroism, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry) and computational approaches. The structure of EI^sc was obtained by homology modeling of the isolated N- and C-terminal domains of other EI proteins. The experimental results indicate that at physiological pH, the dimeric EI^sc had a well-folded structure; however, at low pH, EI^sc showed a partially unfolded state with the features of a molten globule, as suggested by fluorescence, far-ultraviolet circular dichroism, FTIR, and 8-anilino-1-naphthalene-sulfonic acid binding. The thermal stability of EI^sc, in the absence of PEP and Mg²⁺, was maximal at pH 7. The presence of PEP and Mg²⁺ did not change substantially the secondary structure of the protein, as indicated by FTIR measurements. However, quenching experiments and proteolysis patterns suggest conformational changes in the presence of PEP; furthermore, the thermal stability of EI^sc was modified depending on the effector added. Our approach suggests that thermodynamical analysis might reveal subtle conformational changes.

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