Pompea Del Vecchio

Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases

The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements. Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins. Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale. The present results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for the stability of the two esterases.

Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus

Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading OPs are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (PTE) (SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against “quorum sensing signals”, and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic PTEs from Pseudomonas diminuta and Agrobacterium radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers.

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, L( -- ) and D( + ) arabitol, D-mannitol, D-sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (deltadH) and temperature of the maximum of the denaturation peak (Tmax) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of L( -- ) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for L( -- ) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of L( -- ) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.

Thermodynamics of formation of inclusion compounds in water. α-Cyclodextrin–alcohol adducts at 298.15 K

The interaction in water of ethanol, n-propanol, n-butanol, isobutanol, s-butanol and t-butanol with α-cyclodextrin (hexacycloamylose, αCD) has been studied calorimetrically at 298.15 K. The results indicate that, with the exception of the t-butanol, these alcohols form inclusion complexes with this dextrin. The calorimetric method employed allows the determination of the thermodynamic parameters characterizing the binding process, namely the enthalpy, ΔHB°, the apparent equilibrium constant,K′B, and then the free energy, ΔGB°′, and entropy, TΔSB°′. The complexes that result are weak, as shown by the low values of the constants. The absolute values of ΔHB° and K′B increase with increasing length of the alkyl chain. Branching of the alkyl chain lowers the value of K′B. Evidence is given to show that t-butanol does not form a complex with αCD, probably because of steric hindrance: for this system the calorimetric data are treated according to an approach derived from the McMillan–Mayer treatment of solutions.

The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 degrees C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C(2) to C(12) and the preferred substrate was pNP-pentanoate showing a k(cat) = 26.2 +/- 0.1 s(-1), K(M) = 0.122 +/- 0.006 mM and a k(cat)/K(M) = 215 +/- 11 mM(-1) s(-1). The enzyme was strongly inhibited by Hg(2+), Zn(2+), Cu(2+), Fe(3+), Mn(2+) ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C(50) values of 1.98 M and 0.92 M, respectively. The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL. Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of this region in the catalytic process.

Identification and physicochemical characterization of BldR2 from Sulfolobus solfataricus, a novel archaeal member of the MarR transcription factor family.

The multiple antibiotic resistance regulators (MarR) constitute a family of ligand-responsive transcriptional regulators abundantly distributed throughout the bacterial and archaeal domains. Here we describe the identification and characterization of BldR2, as a new member of this family, in the archaeon Sulfolobus solfataricus and report physiological, biochemical, and biophysical investigation of its stability and DNA binding ability. Transcriptional analysis revealed the upregulation of BldR2 expression by aromatic compounds in the late-logarithmic growth phase and allowed the identification of cis-acting sequences. Our results suggest that BldR2 possesses in solution a dimeric structure and a high stability against both temperature and chemical denaturing agents; the protein binds site specifically to its own promoter, Sso1082, with a micromolar binding affinity at two palindromic sites overlapping TATA-BRE and the transcription start site. Benzaldehyde and salicylate, ligands of MarR members, are antagonists of binding of DNA by BldR2. Moreover, two single-point mutants of BldR2, R19A and A65S, properly designed for obtaining information about the dimerization and the DNA binding sites of the protein, have been produced and characterized. The results point out an involvement of BldR2 in the regulation of the stress response to aromatic compounds and point to arginine 19 as a key amino acid involved in protein dimerization, while the introduction of serine 65 increases the DNA affinity of the protein, making it comparable with those of other members of the MarR family.

Dimerisation and structural integrity of Heparin Binding Hemagglutinin A from Mycobacterium tuberculosis: Implications for bacterial agglutination

MINT‐ 7709940 , MINT‐ 7709948 : HBHA (uniprotkb:A5TZK3) and HBHA (uniprotkb:A5TZK3) bind (MI: 0407) by circular dichroism (MI: 0016)MINT‐ 7709966: HBHA (uniprotkb:A5TZK3) and HBHA (uniprotkb:A5TZK3) bind (MI: 0407) by biophysical (MI: 0013)MINT‐ 7709955: HBHA (uniprotkb:A5TZK3) and HBHA (uniprotkb:A5TZK3) bind (MI: 0407) by dynamic light scattering (MI: 0038)

Comprehensive analysis of surface charged residues involved in thermal stability in Alicyclobacillus acidocaldarius esterase 2

Here we report a comprehensive analysis through alanine-scanning mutagenesis of the contribution of surface ion pairs to the thermal stability of Alicyclobacillus acidocaldarius esterase 2 (EST2). We produced 16 single mutants, 4 double mutants corresponding to selected ion pairs R31/E118, E43/K102, R58/D130, D145/R148, 2 double mutants (R63A/R98A and E50A/D94A) involving residues of a large ion network on the protein surface and the double-mutant R98A/R148A meant to disrupt the R98 interactions within the said network and, contextually, the interaction between R148 and D145. The double-mutant E43A/E273K was obtained by chance. All selected residues were replaced with alanine except E91, which was mutated to a glycine and K102, which was changed to a glutamine. All 24 proteins were over-expressed in Escherichia coli, purified and characterized with respect to the main features. Structural stability data were compared with an in silico prediction of ΔΔG values. Our study of the individual factors involved in thermostability and their structural interpretation reveals that the great stability of this thermophilic protein can be explained by the contribution of a few residues at the protein surface.

Excess enthalpies of ternary aqueous solutions of amides and ureas at 298.15 K

Aqueous solutions of amides have been studied for their importance as uncharged model molecules of structural units of polypeptide chains and proteins. Urea and its alkyl derivatives are known as denaturing agents against proteins. In this paper some new experimental calorimetric data from our laboratory concerning ternary aqueous solutions, each containing one amide and one urea species, are presented, discussed and compared with literature data. The relevance of the weak interactions occurring between these types of cosolutes in water is discussed from the point of view of the biochemical consequences on the stability of proteins. It is confirmed that both polar and hydrophobic intermolecular interactions perturb the native conformation of polypeptides and proteins, as they compete with intramolecular interactions. Urea is either ineffective or it enhances the hydrophobic interactions, contrary to what apparently is suggested by the increase of solubility of lighter hydrocarbons in urea–water mixtures.

A new peptide-based fluorescent probe selective for zinc(II) and copper(II)

A novel metal ion-sensitive fluorescent peptidyl-probe has been designed based on the most common five-residue repeat in mammalian histidine rich glycoproteins (HRGs). A dansyl-amide moiety at the N-terminus and a tryptophan residue at the C-terminus of the peptide were added as they can act as a FRET (fluorescence resonance energy transfer) pair. The dansyl fluorophore was chosen also because it frequently shows strong CHEF (chelation enhanced fluorescence) and solvatochromic effects. The designed peptide, dansyl-HPHGHW-NH2 (dH3w), showed a selective fluorescence turn-on response to Zn2+ in aqueous solutions at pH 7.0 when excited at both 295 nm and 340 nm, thus indicating that both FRET and CHEF or solvatochromic effects are active in the metal/peptide complex. Steady-state fluorescence and isothermal titration calorimetry (ITC) measurements demonstrated that two peptide molecules bind to one zinc ion with an association constant Ka = 5.7 × 105 M-1 at 25 °C and pH 7.0. The fluorescence response to Zn2+ was not influenced by Pb2+, Cd2+, Mn2+, Fe2+, Fe3+, Mg2+, Ca2+, K+ and Na+ ions and only slightly influenced by Co2+ and Ni2+. Copper(ii), at concentrations as low as 5 μM, caused a strong quenching of both free and Zn2+ complexed dH3w. The determination of the binding parameters for Cu2+ has shown that one copper ion binds to one dH3w molecule with an association constant of 1.2 × 106 M-1 thus confirming the higher affinity of peptide for Cu2+ than for Zn2+. Finally, we demonstrated that dH3w can penetrate into HeLa cells and could thus be used for the determination of intracellular Zn2+ and Cu2+ concentrations.