Pilar Usobiaga

Structural Characterization of the Unligated and Choline-bound Forms of the Major Pneumococcal Autolysin LytA Amidase CONFORMATIONAL TRANSITIONS INDUCED BY TEMPERATURE

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I′ band, results in 19% of α-helix and tight loops, 47% of β-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.

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.

Conformational changes in bacteriophage Ø 29 connector prevents DNA-binding activity

In vitro DNA packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. Proteolytic cleavage of p10 rendered the proheads inactive for DNA packaging. A similar treatment on isolated connectors abolished the DNA-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. Analytical ultracentrifugation showed that the proteolyzed connectors had a smaller sedimentation coefficient, while amino acid analysis after dialysis of the proteolyzed p10 confirmed the loss of 16 and 19 amino acids from the amino and carboxy termini, respectively. Low angle X-ray scattering revealed that proteolysis was followed by a small decrease in the radius of gyration and a reorganization of the distal domain of the cylindrical inner part of the connector. Characterization of the cleavage sites in the primary sequence allowed us to propose the location of the DNA-binding domain in the connector model.

Biophysical properties of bacteriophage φ29

Abstract Bacteriophage φ29 has been purified in large scale to homogeneity. The sedimentation and diffusion coefficients of the virus are (256 ± 2) × 10−13 sec and (8.8 ± 0.5) × 10−8 cm2 × sec−1, respectively. The value of the partial specific volume of φ29, obtained by pycnometry, is 0.61 ± 0.01 cm3 × g−1. From these values and the Svedberg equation, the molecular mass of φ29 is (18 ± 1) × 106 daltons. From the phosphorus and nitrogen content, and the amino acid and nucleotide composition, the DNA percentage in φ29 is 56 ± 2%. Other independent estimations of the molecular mass of φ29 are given.

Studies on poly(ethyl acrylate) in θ solvents

Abstract The θ temperatures have been determined for solutions of poly(ethyl acrylate) in methyl, ethyl, n-propyl and n-butyl alcohols. Viscometric and sedimentation behaviour of this polymer in n-propanol have been studied. At 28° the following expressions have been obtained: [ƞ] = 3·94.10 −4 . M n 0·52 s 0 = 1·0.10 −2 . M n 0·47

Physicochemical studies on tryptic digestion of bovine fibrinogen

The high molecular weight fragments observed during tryptic digestion of bovine fibrinogen and the variation of their relative proportion with time has been studied. Separation of the different molecular species was carried out by gel filtration and the molecular weights of the isolated fragments were determined by sedimentation equilibrium and from their electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels. The fibrinogen is degraded by trypsin into distinct fragments, with molecular weights of 270 000, 170 000, 90 000 and 50 000 accompanied by a series of smaller fragments whose properties were not investigated. The relative proportion of the components was estimated from area measurements on scans of the stained gels obtained after electrophoresis in the presence of sodium dodecylsulfate. The relative concentration and the molecular weight of each component established its molar concentration in each of the digestion mixtures obtained after varying incubation times (1-60 min). These data were used for a kinetic analysis of the process. The kinetic model derived on the basis of the trinodular model of fibrinogen (see Appendix) gave a very good representation of all the experimental results.