José Laynez

Analysis of the solvent effect on the photophysics properties of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN).

The absorption and emission spectroscopic properties of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) have been studied in a large number of protogenic, nonprotogenic, and amphiprotic solvents. The data obtained can be explained by the inclussion of a new term in the Lippert equation which takes into account the acidity of the solvent. This finding indicates that some precaution should be taken when using PRODAN as an indicator of the polarity of protein cavities if the environments involved include acid sites.

NMR investigations of protein-carbohydrate interactions : Studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N ',N ''-triacetylchitotriose

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N′,N′′‐triacetylchitotriose has been analyzed by 1H‐NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution‐structure determination by NOESY NMR experiments in water. The NMR‐analysis was extended to assessment of the topology of the complex between pseudohevein and N,N′,N′′‐triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton‐proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand‐induced conformational change. The average backbone (residues 3‐41) RMSD of the 20 refined structures was 1.14 Å, whereas the heavy atom RMSD was 2.18 Å. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectins capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr‐substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin‐1 and a mammalian C‐type lectin. A comparison of the three‐dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA‐B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems. Proteins 2000;40:218–236.

Structural and thermodynamic characterization of Pal, a phage natural chimeric lysin active against pneumococci.

Pal amidase, encoded by pneumococcal bacteriophage Dp-1, represents one step beyond in the modular evolution of pneumococcal murein hydrolases. It exhibits the choline-binding module attaching pneumococcal lysins to the cell wall, but the catalytic module is different from those present in the amidases coded by the host or other pneumococcal phages. Pal is also an effective antimicrobial agent against Streptococcus pneumoniae that may constitute an alternative to antibiotic prophylaxis. The structural implications of Pal singular structure and their effect on the choline-amidase interactions have been examined by means of several techniques. Pal stability is maximum around pH 8.0 (Tm ≅ 50.2 °C; ΔHt = 183 ± 4 kcal mol–1), and its constituting modules fold as two tight interacting cooperative units whose denaturation merges into a single process in the free amidase but may proceed as two well resolved events in the choline-bound state. Choline titration curves reflect low energy ligand-protein interactions and are compatible with two sets of sites. Choline binding strongly stabilizes the cell wall binding module, and the conformational stabilization is transmitted to the catalytic region. Moreover, the high proportion of aggregates formed by the unbound amidase together with choline preferential interaction with Pal dimers suggest the existence of marginally stable regions that would become stabilized through choline-protein interactions without significantly modifying Pal secondary structure. This structural rearrangement may underlie in vitro “conversion” of Pal from the low to the full activity form triggered by choline. The Pal catalytic module secondary structure could denote folding conservation within pneumococcal lytic amidases, but the number of functional choline binding sites is reduced (2–3 sites per monomer) when compared with pneumococcal LytA amidase (4–5 sites per monomer) and displays different intermodular interactions.

Effect of the antitumour protein α-sarcin on the thermotropic behaviour of acid phospholipid vesicles

The antitumour protein alpha-sarcin modifies the thermotropic behaviour of phospholipid vesicles. This has been studied by fluorescence depolarization measurements and differential scanning calorimetry. A surface protein-phospholipid interaction is detected by measuring the polarization degree of TMA-DPH-labelled vesicles. At the higher protein/lipid molar ratios studied, the alpha-sarcin-vesicles complexes exhibit different thermotropic behaviour depending on whether they are prepared above or below the Tm of the corresponding phospholipid. Labelling of the protein with photoactive phospholipids has also been considered. alpha-Sarcin penetrates the bilayer deep enough to be labelled with the photoactive group located at the C-12 of the fatty acid acyl chain of phospholipids forming vesicles.

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier‐transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide 1 infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist‐induced alterations by band‐fitting analysis of the amide 1 spectral band reveals no appreciable changes in the percent of α‐helix, but a decrease in β‐sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration‐dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinerpic agonist implies significant rearrangements in the protein structure.

Characterization of Ejl, the cell‐wall amidase coded by the pneumococcal bacteriophage Ej‐1

The Ejl amidase is coded by Ej‐1, a temperate phage isolated from the atypical pneumococcus strain 101/87. Like all the pneumococcal cell‐wall lysins, Ejl has a bimodular organization; the catalytic region is located in the N‐terminal module, and the C‐terminal module attaches the enzyme to the choline residues of the pneumococcal cell wall. The structural features of the Ejl amidase, its interaction with choline, and the structural changes accompanying the ligand binding have been characterized by CD and IR spectroscopies, differential scanning calorimetry, analytical ultracentrifugation, and FPLC. According to prediction and spectroscopic (CD and IR) results, Ejl would be composed of short β‐strands (ca. 36%) connected by long loops (ca. 17%), presenting only two well‐predicted α‐helices (ca. 12%) in the catalytic module. Its polypeptide chain folds into two cooperative domains, corresponding to the N‐ and C‐terminal modules, and exhibits a monomer ↔ dimer self‐association equilibrium. Choline binding induces small rearrangements in Ejl secondary structure but enhances the amidase self‐association by preferential binding to Ejl dimers and tetramers. Comparison of LytA, the major pneumococcal amidase, with Ejl shows that the sequence differences (15% divergence) strongly influence the amidase stability, the organization of the catalytic module in cooperative domains, and the self‐association state induced by choline. Moreover, the ligand affinity for the choline‐binding locus involved in regulation of the amidase dimerization is reduced by a factor of 10 in Ejl. Present results evidence that sequence differences resulting from the natural variability found in the cell wall amidases coded by pneumococcus and its bacteriophages may significantly alter the protein structure and its attachment to the cell wall.

Iminophosphorane-substituted proton sponges. Part 4. Comparison of X-ray molecular structures with solution properties (pKa, 1H and 13C NMR spectroscopy)

Classical ‘proton sponges’ related to 1,8-bis(dimethylamino) naphthalene (DMAN) and new ones derived from the replacement of one or both dimethylamino groups by iminophosphorane groups are compared (i) in the solid state using crystallographic data, (ii) in solution in deuteriochloroform and [2H6]dimethyl sulfoxide by 1H and 13C NMR spectroscopy, (iii) in water and ethanol–water mixtures by determining their pKa′ s. The conclusion is that the new sponges are stronger than the classical ones but, since they decompose when the acid proton is removed are of no practical value as chemical reagents.

Towards a solvent acidity scale: the calorimetry of the N-methyl imidazole probe

This paper reports a rather straightforward calorimetric method for the precise determination of the acidity of organic solvents. From the calculated enthalpies of solvation (ΔHsolv) of the probe compounds N-methylimidazole and N-methylpyrrole and the known relative permittivity (Iµ) of the solvent ΔHacid is obtained through the equation: ΔHacid=–[ΔH0solv(N-methylimidazole)-ΔH0solv(N-methylpyrrole)]+ 18.760 f(Iµ)+ 1.69. The proposed method allowed us to determine the acidity of 36 solvents, including some slightly acidic ones, whose acidity is difficult to obtain by existing methods.

Structural Domain Organization of Gastric H+,K+-ATPase and Its Rearrangement during the Catalytic Cycle

Differential scanning calorimetry has been used to characterized the thermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacity function of (H+,K+)-ATPase in highly oriented gastric vesicles displays two peaks at 53.9°C (Tm1) and 61.8°C (Tm2). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state processes. They can be assigned to two cooperative domains located in the cytoplasmic loops of the α-subunit, according to the disappearance of the endothermic signal upon removal of these regions by proteinase K digestion. Analysis of the thermal-induced unfolding of the enzyme trapped in different catalytic cycle intermediates has allowed us to get insight into the E1-E2 conformational change. In the E1 forms both transitions are always observed. As Tm1 is shifted to Tm2 by vanadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions. Stabilization of the E2 forms results in the disappearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in Tm2 and ΔH1. The catalytic domain melts following a process in which intermolecular interactions either in the native or in the unfolded state might be involved. Interestingly, the E2-vanadate-K+ form displays intermediate properties between the E1 and E2 conformational families.

CALORIMETRIC QUANTIFICATION OF THE HYDROGEN-BOND ACIDITY OF SOLVENTS AND ITS RELATIONSHIP WITH SOLVENT POLARITY

A new solvent polarity–polarizability scale (SPP) has been used to reevaluate the hydrogen-bond acidity scale of organic solvents previously reported and has been extended to a new set of solvents. The hydrogen-bond acidity, expressed as the enthalpy term ΔacidH, has been evaluated by measuring the differences between the solvation enthalpies of N-methylimidazole and N-methylpyrrole in these solvents along with the solvent polarity–polarizability (SPP) values. The ΔacidH values for 63 solvents are reported.