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The bacterial phosphoenolpyruvate-dependent sugar phosphotransferase system is a multiprotein complex that phosphorylates and, concomitantly, transports carbohydrates across the membrane into the cell. The first protein of the cascade is a multidomain protein so-called enzyme I (EI). The N-terminal domain of EI from Streptomyces coelicolor, EIN(sc), responsible for the binding to the second protein in the cascade (the histidine phosphocarrier, HPr), was cloned and successfully expressed and purified. We have previously shown that EI(sc) binds to HPr(sc) with smaller affinity than other members of the EI and HPr families [Hurtado-Gómez et al. (2008) Biophys. J., 95, 1336-1348]. We think that the study of the isolated binding HPr(sc) domain, that is EIN(sc), could shed light on the small affinity value measured. Therefore, in this work we present a detailed description of the structural features of the EIN domain, as a first step towards a complete characterization of the molecular recognition process between the two proteins. We show that EIN(sc) is a folded protein, with alpha-helix and beta-sheet structures and also random-coil conformations, as shown by circular dichroism (CD), FTIR and NMR spectroscopies. The acquisition of secondary and tertiary structures, and the burial of hydrophobic regions, occurred concomitantly at acidic pHs, but at very low pH, the domain acquired a molten-globule conformation. The EIN(sc) protein was not very stable, with an apparent conformational free energy change upon unfolding, DeltaG, of 4.1 +/- 0.4 kcal mol(-1), which was pH independent in the range explored (from pH 6.0 to 8.5). The thermal denaturation midpoint, which was also pH invariant, was similar to that measured in the isolated intact EI(sc). Although EIN(sc) shows thermal- and chemical denaturations that seems to follow a two-state mechanism, there is evidence of residual structure in the chemical and thermally unfolded states, as indicated by differential scanning calorimetry and CD measurements.

protein engineering, design and selection

The N-terminal domain of the enzyme I is a monomeric well-folded protein with a low conformational stability and residual structure in the unfolded state