Biomedical subjects
Publications and source records attributed to Giuseppe Graziano.
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It has been noted that the work of cavity creation in water exhibits a crossover behavior, in that its cavity size dependence changes from volume dependence for small cavities to area dependence for larger cavities [Lum, K.; Chandler, D.; Weeks, J. D. J. Phys. Chem. B 1999, 103, 4570]. It is shown here that this behavior can be reproduced using the scaled particle theory in a straightforward manner for six different liquids (water, methanol, ethanol, benzene, cyclohexane, and carbon tetrachloride). It has also been suggested that the crossover is due to a change in the physical mechanism of the process, from one entropy-dominated to another enthalpy-dominated. However, the crossover behavior can be produced using the scaled particle theory without invoking any change in any physical mechanism. Also, the crossover occurs at a length scale of the size of the liquid molecules, as has been pointed out by others. This is the length regime where the work of cavity creation bears little relation to the bulk liquid surface tension. In addition, it is pointed out that cavity creation can always be considered as a purely entropy-driven process, which is usually accompanied by another process with compensating enthalpy and entropy changes.
The partial molar volume of n-alcohols at infinite dilution in water is smaller than the molar volume in the neat liquid phase. It is shown that the formula for the partial molar volume at infinite dilution obtained from the scaled particle theory equation of state for binary hard sphere mixtures is able to reproduce in a satisfactory manner the experimental data over a large temperature range. This finding implies that the packing effects play the fundamental role in determining the partial molar volume at infinite dilution in water also for solutes, such as n-alcohols, forming H bonds with water molecules. Since the packing effects in water are largely related to the small size of its molecules, the latter feature is the ultimate cause of the decrease in partial molar volume associated with the hydrophobic effect.
The stability against chemical denaturants of the elongation factor EF-1alpha (SsEF-1alpha), a protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus has been characterized in detail. Indeed, the atypical shape of the protein structure and the unusual living conditions of the host organism prompted us to analyze the effect of urea and guanidine hydrochloride (GuHCl) on the GDP complex of the enzyme (SsEF-1alpha x GDP) by fluorescence and circular dichroism. These studies were also extended to the nucleotide-free form of the protein (nfSsEF-1alpha). Interestingly, the experiments show that the denaturation curves of both SsEF-1alpha forms present a single inflection point, which is indicative of a cooperative unfolding process with no intermediate species. Moreover, the chemically induced unfolding process of both SsEF-1alpha x GDP and nfSsEF-1alpha is fully reversible. Both SsEF-1alpha forms exhibit remarkable stability against urea, but they do not display a strong resistance to the denaturing action of GuHCl. These findings suggest that electrostatic interactions significantly contribute to SsEF-1alpha stability.
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Computer simulations [S. Shimizu and H. S. Chan, J. Am. Chem. Soc. 123, 2083 (2001); D. Paschek, J. Chem. Phys. 120, 10605 (2004)] have demonstrated that the heat-capacity change associated with the interaction of two nonpolar spherical particles, at room temperature, shows a complex behavior with a significant maximum at the distance corresponding to the desolvation barrier configuration and a small minimum at the distance corresponding to the contact configuration. Taking advantage of the detailed analysis performed by Paschek, the two-state model of Muller is applied to estimate the energetic strength and the intactness of the H bonds in the hydration shell of a xenon atom and in the concave part of the joint Xe-Xe hydration shell. In both hydration shell regions the H bonds are energetically stronger but more broken than those in bulk water. In addition, those in the concave part of the joint Xe-Xe hydration shell are, in absolute, stronger and more broken. These thermodynamic features coupled to simple geometric arguments allow the calculation of heat-capacity values that are in agreement with those provided by computer simulations for the pairwise Xe-Xe interaction.
Using experimental data from the literature, entropy convergence in the hydration thermodynamics of n-alcohols is shown to occur at about 125 degrees C. The phenomenon is reproduced in a more-than-qualitative manner by means of a theoretical approach that accounts for the entropy contributions associated with (a) creation of a cavity in water, (b) turning on solute-water van der Waals interactions, and (c) turning on the solute-water H-bonding potential. The density of water and the effective size of water molecules with their temperature dependence play the pivotal role for the occurrence of entropy convergence, together with the property of the alcohol hydroxyl group to form the same number of H-bonds with water molecules regardless of the length of the alkyl chain.
Angell developed a simple two-state model of hydrogen bonds with the aim to describe some properties of pure water. Muller extended the two-state model description to treat the unusual thermodynamics of hydrophobic hydration. We show here that, to correctly reproduce a qualitative feature of the temperature dependence of the hydration heat capacity change of nonpolar solutes by means of the two-state Muller's model, the hydrogen bonds in the hydration shell have to be more broken than those in bulk water. This contrasts with the suggestion in the literature that more hydrogen bonds form around a nonpolar solute in water.
The heat capacity change associated with the hydration of benzene is a large and positive quantity, but it is significantly smaller than that associated with the hydration of an alkane having the same accessible surface area of benzene, the corresponding alkane. This large difference merits attention and should be rationalized. This task is performed by means of the two-state Muller's model for the reorganization of H-bonds. It results that: (a) the hydration shell of both hydrocarbons consists of H-bonds that are enthalpically stronger but slightly more broken than those in bulk water; (b) the hydration shell of benzene consists, on average, of enthalpically slightly weaker H-bonds with respect to the corresponding alkane. The latter feature, due to the presence of the weak benzene-water H-bonds, is the physical cause of the large difference in the hydration heat capacity change, according to the two-state Muller's model.
The thermal stability of the two dimers of RNase A with N- or C-terminal swapped ends is investigated by means of dissociation kinetics, differential scanning calorimetry, and circular dichroism measurements. The data indicate that the dimer characterized by the swapping of the N-terminal alpha-helices is less prone to monomerize when compared to the dimer characterized by the swapping of the C-terminal beta-strands. This finding is correlated to the structural features of the so-called open interface of the dimeric forms.
S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.
Experimental data for the solvation of water in nonpolar organic solvents indicate that the process is spontaneous under the Ben-Naim standard conditions, due to a large and negative enthalpy change. The process is analyzed by considering that the solvation Gibbs energy change is given by the sum of two opposing terms: the work to create a suitable cavity and the work to turn on the attractive solute-solvent interactions. Basic calculations point out unequivocally that, beyond the van der Waals contributions, additional favorable interactions occur between water and the surrounding solvent molecules. These additional favorable interactions should be nontraditional hydrogen bonds such as those between the delocalized pi-electron cloud of the aromatic ring and the hydrogen atoms of water, and those between the CH groups of both aliphatics and aromatics and the oxygen atom of water.
Sso7d is a 62-residue protein from the hyperthemophilic archaeon Sulfolobus solfataricus with a denaturation temperature close to 100 degrees C around neutral pH. An engineered form of Sso7d truncated at leucine 54 (L54Delta) is significantly less stable, with a denaturation temperature of 53 degrees C. Molecular dynamics (MD) studies of Sso7d and its truncated form at two different temperatures have been performed. The results of the MD simulations at 300 K indicate that: (1) the flexibility of Sso7d chain at 300 K agrees with that detected from X-ray and NMR structural studies; (2) L54Delta remains stable in the native folded conformation and possesses an overall dynamic behavior similar to that of the parent protein. MD simulations performed at 500 K, 10 ns long, indicate that, while Sso7d is in-silico resistant to high temperature, the truncated variant partially unfolds, revealing the early phases of the thermal unfolding pathway of the protein. Analysis of the trajectories of L54Delta suggests that the unzipping of the N-terminal and C-terminal beta-strands should be the first event of the unfolding pathway, and points out the regions more resistant to thermal unfolding. These findings allow one to understand the role played by specific interactions connecting the two ends of the chain for the high thermal stability of Sso7d, and support recent hypotheses on its folding mechanism emerged from site-directed mutagenesis studies.
The stability of acetyl-esterase, Aes, from Escherichia coli against the denaturing action of urea and guanidine hydrochloride, GuHCl, has been investigated by means of circular dichroism and fluorescence measurements. The urea-induced unfolding curves show a single inflection point at 6.2 M urea, whereas the GuHCl-induced curves show two inflection points at 1.4 and 3.1 M GuHCl. The unfolding process is reversible with both urea and GuHCl. These results, together with similar experimental data on the mutant form V20D-Aes, suggest the presence of two domains in the Aes structure, which unfold more or less independently depending on the denaturant used. This is also supported by a 3D model obtained by homology modeling using the structure of brefeldine as a template. The effect of NaCl on the urea-induced unfolding curves of the enzyme has also been investigated.
By comparing the hydration thermodynamics of benzene with that of a hypothetical aliphatic hydrocarbon having the same accessible surface area (ASA) of benzene, Makhatadze and Privalov concluded that the whole difference is due to the weak H-bonds that water forms with the aromatic ring. The formation of such H-bonds would be characterized by a negative Gibbs energy change, slightly increasing in magnitude with temperature, and a positive entropy change over a large temperature range. The latter thermodynamic feature is not physically reliable for the formation of H-bonds. In the present article, by using a statistical mechanical dissection scheme of hydration, a microscopic interpretation for the numbers obtained by Makhatadze and Privalov is proposed. The difference in hydration Gibbs energy should be attributed to the different strength of van der Waals interactions that benzene can do with water, owing to the larger polarizability of the aromatic ring with respect to an aliphatic hydrocarbon of equal size. In addition, the difference in hydration entropy should account for the different extent of H-bond reorganization upon the insertion of benzene and the corresponding aliphatic hydrocarbon in water.
It has been recently claimed that the large cohesive energy density of water is the ultimate cause of the poor solubility of nonpolar compounds in water. In order to test the validity of this idea, we analyze the difference in solubility between light water and heavy water of several nonpolar compounds. Even though the cohesive energy density of D(2)O is larger than that of H(2)O, nonpolar compounds are slightly more soluble in D(2)O than H(2)O. In such case experimental data do not support the correctness of the large cohesive energy density as the ultimate cause of hydrophobicity. We show that D(2)O is a slightly better solvent than H(2)O for nonpolar compounds because it is slightly less costly to create a cavity in the former liquid. This is because there is slightly more void volume in heavy water than in light water.