Why are there so many dimeric proteins and enzymes? While for heterodimers a functional explanation seems quite reasonable, the case of homodimers is more puzzling. The number of homodimers found in all living organisms is rapidly increasing. A thorough inspection of the structural data from the available literature and stability (measured from denaturation–renaturation experiments) allows one to suggest that homodimers can be divided into three main types according to their mass and the presence of a (relatively) stable monomeric intermediate in the folding–unfolding pathway. Among other explanations, we propose that an essential advantage for a protein being dimeric may be the proper and rapid assembly in the cellular milieu.
Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca(2+) and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca(2+), the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca(2+)) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximately 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.
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 ...
Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min−1 for the inhibitor-bound enzyme to 0.12±0.02 min−1 for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.
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