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

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...

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Bibliographic Details
Published in:Protein engineering, design and selection design and selection, 2010-09, Vol.23 (9), p.729-742
Main Authors: Romero-Beviar, Manuel, Martínez-Rodríguez, Sergio, Prieto, Jesús, Goormaghtigh, Erik, Ariz, Usue, Martínez-Chantar, María de la Luz, Gómez, Javier, Neira, José L.
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Circular Dichroism
Enzyme Stability
Escherichia coli - genetics
Hydrogen-Ion Concentration
Hydrophobic and Hydrophilic Interactions
N-terminal domain
Nuclear Magnetic Resonance, Biomolecular
Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry
Phosphoenolpyruvate Sugar Phosphotransferase System - genetics
Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism
Phosphotransferases (Nitrogenous Group Acceptor) - chemistry
Phosphotransferases (Nitrogenous Group Acceptor) - genetics
Phosphotransferases (Nitrogenous Group Acceptor) - metabolism
Protein Conformation
Protein Denaturation
Protein Folding
protein stability
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
spectroscopy
Spectroscopy, Fourier Transform Infrared
Streptomyces coelicolor
Streptomyces coelicolor - enzymology
Streptomyces coelicolor - genetics
structure
Thermodynamics
unfolded state
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Summary: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, EINsc, 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 EIsc binds to HPrsc 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 HPrsc domain, that is EINsc, 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 EINsc is a folded protein, with α-helix and β-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 EINsc protein was not very stable, with an apparent conformational free energy change upon unfolding, ΔG, 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 EIsc. Although EINsc 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.
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzq045