The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier protein. Characterization of cold denaturation and urea-protein interactions.
Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 +/- 0.3 kcal.mol(-1).K(-1), at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 +/- 4 kcal.mol(-1), the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m(DeltaH(i) )), entropy (m(DeltaS(i) )) and heat capacity (m(DeltaC(pi) )), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m(DeltaH(i) ) and the m(DeltaS(i) ) are negative at low temperatures, but as the temperature is increased, m(DeltaH(i) ) makes a less favourable contribution than m(DeltaS(i) ) to the change in free energy upon urea binding. The m(DeltaC(pi) ) is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small.
Neira
José L
JL
Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, Avda. del Ferrocarril s/n, 03202 Elche (Alicante), Spain. jlneira@umh.es
Gómez
Javier
J
D001426:Bacterial Proteins / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003080:Cold Temperature; D004795:Enzyme Stability; D005453:Fluorescence; D006358:Hot Temperature; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D013302:Streptomyces; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D014508:Urea; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr