Biophysical characterization of the Enzyme I of the Streptomyces coelicolor phosphoenolpyruvate: sugar phosphotransferase system

¹ Instituto de Biologia Molecular y Celular
² Friedrich-Alexander Universitat
³ University of Miguel Hernandez

^* To whom correspondence should be addressed. E-mail: jlneira{at}umh.es.

Submitted on October 31, 2005
Revised on November 28, 2005
Accepted on 7 March 2006

	Abstract

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-UV CD, FTIR and DSC) and computational approaches. The structure of EI^sc was obtained by homology modelling 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-UV CD, FTIR and ANS-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 report subtle conformational changes.

Key Words: Circular dichroism, FTIR, homology modelling, molten-globule, protein folding, protein stability

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