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The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is ΔG = 4.0 ± 0.2 kcal·mol−1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7–9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5–4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family.

We have analysed the hydrogen/deuterium exchange of the tetramerization domain of human tumour suppressor p53 under mild chemical denaturation conditions, and at different temperatures. Exchange behaviour has been measured for 16 amide protons in the chemical-denaturation studies and for seven protons in the temperature-denaturation studies. The exchange rates are in the range observed for other proteins with similar elements of secondary structure. The slowest-exchange core includes contributions from residues in the alpha helix and the beta sheet. However, only some of the slowest-exchanging protons correspond to residues involved in native interactions in the transient intermediate detected during the folding of this domain. The guanidinium-chloride denaturation curves of all residues seem to merge together, although they are well below the main isotherm of global unfolding. Thus, there is no evidence for several subglobal unfolding units. The activation parameters obtained from the temperature-denaturation experiments are similar to those obtained for monomeric proteins, and well below the global unfolding enthalpy obtained by circular dichroism measurements. Thus, the exchange studies at different denaturant concentrations and temperatures indicate that no particular folding intermediate is populated under those conditions.

Native-like complexes of proteins, formed by the association of two complementary fragments comprising the entire sequence of the protein, can be used to gain insight into the stability and folding of the intact protein. We have studied the structural, thermodynamic and kinetic properties of four barnase complexes, with the cleavage site at different positions of the amino-acid chain (CB36, at position 36; CB56, at position 56; CB68, at position 68; and CB79, at position 79). The four barnase complexes have native-like structure as shown by fluorescence, far-and near-UV CD, size-exclusion chromatography and NMR. The NMR characterization indicated that the structural changes were mainly located in regions close to the cleavage site. The main core of the protein was fully formed and the overall structure was similar to that of intact barnase. The thermal and chemical denaturation showed that all complexes were substantially destabilized. CB56 displayed two denaturation transitions, probably because of the presence of partially folded conformations around the cleavage site. The rate constant for the association/folding of fragments decreased with the decreasing length of the C-terminal fragment. Thus, the larger the fragment (and, consequently, the larger the amount of residual native-like structure), the faster the association. These findings are consistent with the proposed model of barnase folding.

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.

The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 degrees C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is deltaG = 4.0 +/- 0.2 kcal x mol-1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7-9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5-4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 +/- 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family.

Assignments for 1H-NMR resonances of most of the residues of bovine pancreatic ribonuclease (RNase A) have been obtained by sequence-specific methods. Identification and classification of spin systems have been carried out by two-dimensional phase-sensitive correlated spectroscopy (360 MHz) and single relayed coherence transfer spectroscopy. Sequence-specific assignments have been achieved by phase-sensitive two-dimensional nuclear Overhauser effect spectroscopy. To overcome the problem of spectral overlap use has been made of (a) an exhaustive analysis of partly exchanged RNase A (spectra in D2O), (b) a comparison with the subtilisin-modified enzyme (RNase S) and (c) small spectral perturbations caused by changes in pH and temperature. The secondary structure elements have been identified from the observed sequential, medium and long-range nuclear Overhauser effects together with data from amide-exchange rates. All information collected leads to the conclusion that the crystal and the solution structures are closely similar.