The conformational stability of theStreptomyces coelicolorhistidine‐phosphocarrier protein

Abstract

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 lowtemperature 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. Colddenaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 ± 0.3 kcalAEmol )1 AEK )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 kcalAEmol )1 , the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m DH i ), entropy (m DS i ) and heat capacity (m DC pi ), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m DH i and the m DS i are negative at low temperatures, but as the temperature is increased, m DH i makes a less favourable contribution than m DS i to the change in free energy upon urea binding. The m DC pi is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small.Keywords: calorimetry; denaturant-binding interactions; histidine-phosphocarrier; protein stability.A full understanding of the physical interactions underlying the structure, folding and the function of a protein requires a detailed description of its conformational stability in terms of the free energy of unfolding. Such a thermodynamic description relies on the quantitative analysis of denaturant-induced or thermally induced folding-unfolding transitions, measured either spectroscopically or calorimetrically. In both cases, data analyses involves the extrapolation of the thermodynamic parameters to standard conditions, usually 298 K in the absence of denaturant. To extrapolate thermal denaturation data, the change in DC p , and its temperature dependence must be known [1,2]. The extrapolation of data from chemical-denaturation [with either urea or guanidinium hydrochloride (Gdm Cl) as denaturants] is carried out using either the linear free energy model, LEM [3][4][5], or the binding...

Defining the Epitope Region of a Peptide from the Streptomyces coelicolor Phosphoenolpyruvate:Sugar Phosphotransferase System Able to Bind to the Enzyme I

“…The stability and the structure of the His-tagged protein are the same as those of the cleaved protein (10). Protein was purified as described (9).…”

“…There is a large homology between these PTS proteins and those of other antibiotic-resistant species, such as Vibrio cholerae and Streptococcus pneumoniae (3,7,8). We have undertaken an extensive description of the structures and conformational stabilities of the HPr sc and EI sc proteins (9)(10)(11)(12). HPr sc is a single polypeptide chain of 93 amino acids; it lacks cysteine and tyrosine residues, and it has one tryptophan and one phenylalanine.…”

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

“…We have undertaken an extensive description of the structures and conformational stabilities of the PTS proteins of S. coelicolor, first, to understand in depth the mechanism of the PTS; and, second, to provide new clues on the protein folding problem. For instance, we have shown that the thermodynamic folding parameters (DC p and m-values) of the HPr of S. coelicolor, HPr sc , are different from those of the other members of the HPr family (17)(18)(19). Interestingly enough, HPr sc , in contrast to the HPrs in Gram-negative bacteria and low-G1C Gram-positive bacteria (1), is not involved in general carbon regulation (20,21).…”

The Helical Structure Propensity in the First Helix of the Histidine Phosphocarrier Protein of Streptomyces coelicolor

“…The presence of the different components of the PTS in S. coelicolor has been reported, and the corresponding proteins cloned and expressed [6,7]. We have undertaken an extensive description of the structures and conformational stabilities of the HPr and EI proteins in S. coelicolor [8][9][10][11]. HPr contains 93 amino acid residues; it lacks cysteine and tyrosine residues, and it only contains one tryptophan and one phenylalanine residues, close to the active site histidine.…”