The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4^0.4 kcal´mol 21 , while a lower value (6.4^0.4 kcal´mol 21 ) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar.Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8^0.3 kcal´mol 21 and 4.8^0.3 kcal´mol 21 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be 224 mL´mol 21 for Ile7Ser and 255 mL´mol 21 for Phe110Ser.These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.Keywords: azurin; protein folding; hydrophobic interaction; dynamic fluorescence; high pressure.The stability of small globular proteins is a problem of outmost interest in biochemistry and biophysics, as demonstrated by the large literature on this subject. In fact, unlike oligomeric enzymes, which may be formed by several subunits and domains, the simpler structure of small proteins allows both accurate computer simulations and detailed experiments about the main driving forces in the assembly of native molecules. Several spectroscopic techniques (NMR, circular dichroism, fluorescence, etc.) have been exploited to investigate this subject. Measurements under various conditions (temperature, ionic strength, pH or concentration of denaturing agents) have been performed. These studies have produced a huge amount of data about the conformational stability of small proteins, allowing to calculate important parameters like the free energy of unfolding and to detect possible intermediates along the folding process. In the last decade the effect of another physical parameter, i.e. hydrostatic pressure, has become accessible to investigation. Protein unfolding by pressure occurs under isothermal conditions and allows to determine the internal packing density and compressibility of these molecules. However, while for oligomeric enzymes dissociation and other conformational ...
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