JoséG. Gavilanes

Sea Anemone Actinoporins: The Transition from a Folded Soluble State to a Functionally Active Membrane-Bound Oligomeric Pore

Actinoporins are a family of 20-kDa, basic proteins isolated from sea anemones, whose activity is inhibited by preincubation with sphingomyelin. They are produced in monomeric soluble form but, when binding to the plasma membrane, they oligomerize to produce functional pores which result in cell lysis. Equinatoxin II (EqtII) from Actinia equina and Sticholysin II (StnII) from Stichodactyla helianthus are the actinoporins that have been studied in more detail. Both proteins display a beta-sandwich fold composed of 10 beta-strands flanked on each side by two short alpha-helices. Two-dimensional crystallization on lipid monolayers has allowed the determination of low-resolution models of tetrameric structures distinct from the pore. However, the actual structure of the pore is not known yet. Wild-type EqtII and StnII, as well as a nice collection of natural and artificially made variants of both proteins, have been produced in Escherichia coli and purified. Their characterization has allowed the proposal of a model for the mechanism of pore formation. Four regions of the actinoporins structure seem to play an important role. First, a phosphocholine-binding site and a cluster of exposed aromatic residues, together with a basic region, would be involved in the initial interaction with the membrane, whereas the amphipathic N-terminal region would be essential for oligomerization and pore formation. Accordingly, the model states that pore formation would proceed in at least four steps: Monomer binding to the membrane interface, assembly of four monomers, and at least two distinct conformational changes driving to the final formation of the functional pore.

Conformational study of the antitumor protein α-sarcin

Abstract The antitmnor protein α-sarcin is a single polypeptide chain produced by the mold Aspergillus giganteus. It inhibits protein synthesis in some tumor cells by inactivating the larger ribosomal subunit. The secondary structure of the molecule has been studied by circular dichroism and predictive methods. The protein contains about 40% of periodic structures, mainly located at both extremes of the polypeptide chain, β-Turns and aperiodic conformation appear at the central part of the molecule. Two different tyrosine populations have been observed in α-sarcin. Attempts to correlate solvent accessibility and particular protein regions have been carried out by using CD in the near-ultraviolet region, fluorescence and absorbance spectroscopies as well as acrylamide quenching and hydropathy profiles. Five different pH-induced conformational transitions are detected. Two of them, at pH 2.5 and 10.2, are denaturing transitions. These results are explained in terms of the structural features of this molecule, and related to its ribonucleolytic activity and ability to cross cell membranes.

Structural basis for the catalytic mechanism and substrate specificity of the ribonuclease α-sarcin

α‐Sarcin is a ribosome‐inactivating protein which selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA. The solution structure of α‐sarcin has been determined on the basis of 1898 distance and angular experimental constraints from NMR spectroscopy. It reveals a catalytic mechanism analogous to that of the T1 family of ribonucleases while its exquisite specificity resides in the contacts provided by its distinctive loops.

Acid phospholipid vesicles produce conformational changes on the antitumour protein α-sarcin

The antitumour protein alpha-sarcin interacts at neutral pH with acid phospholipid vesicles promoting their aggregation and fusion. This interaction produces conformational changes on the protein molecule. Circular dichroism and infrared spectroscopy have been used to analyze the secondary structure of the protein molecule. The obtained results show an increased alpha-helix content upon interaction with the lipid vesicles. Detergents and halogenated alcohols have also been considered as an approach to the study of the conformational changes produced upon alpha-sarcin-phospholipid vesicles interaction. SDS treatment as well as trifluoroethanol also increase the helical content of alpha-sarcin. Intrinsic fluorescence of the protein has also been measured for the analysis of the conformational changes produced. The above helicogenic treatments produce a decrease on the structural quenching in alpha-sarcin which is consistent with the existence of hydrophobic protein-lipid interactions. The observed conformational changes are interpreted in terms of a shielding from polar groups caused by the lipids, which promotes intrachain hydrogen bonding and decreased static quenching. The reported conformational changes are discussed in terms of electrostatic and hydrophobic interactions involved in the fusion of lipid vesicles promoted by alpha-sarcin, and potentially in the passage of the protein across membrane cells.

Molecular and spectroscopic characterisation of a low molecular weight seed storage protein from yellow mustard (Sinapis alba L.)

Abstract 1. 1. A 1.7S protein has been purified from mustard seeds ( Sinapis alba L.). This protein, soluble in water and dilute salt solutions, is considered as an albumin and constitutes about 10% of the total soluble protein in mustard seeds. 2. 2. Its molecular weight is approximately 15,000 and is composed of two polypeptide chains (M r = 9500 and 5000), linked by two disulfide bridges. 3. 3. The amino acid compositions of both subunits as well as of the native protein are reported, showing a strong homology with napins from Brassica napus L. 4. 4. The ultraviolet absorption, fluorescence emission and circular dichroism spectra of the purified protein have been obtained. The mustard protein exhibits about 50% α-helix with a very low β-structure content. Based on its structural characteristics, a zein-like packing is proposed for this protein from mustard seeds.

MEMBRANE INTERACTION OF A BETA -STRUCTURE-FORMING SYNTHETIC PEPTIDE COMPRISING THE 116-139TH SEQUENCE REGION OF THE CYTOTOXIC PROTEIN ALPHA -SARCIN

alpha-Sarcin is a cytotoxic protein that strongly interacts with acid phospholipid vesicles. This interaction exhibits a hydrophobic component although alpha-sarcin is a highly polar protein. A peptide comprising the amino acid sequence corresponding to the 116-139th segment of the alpha-sarcin cytotoxin has been synthesized by a standard fluoren-9-yl-methoxycarbonyl-based solid phase method. Its primary structure is: (116)-NPGPARVIYTYPNKVFCGIIAHTK-(139). Two beta-strands have been predicted in this region of alpha-sarcin, where the less polar stretches of the protein are found. The synthetic peptide interacts with negatively charged large unilamellar vesicles of either natural or synthetic phospholipids. An apparent fragmentation of the vesicles is produced by the peptide based on electron microscopy studies. The peptide promotes leakage of the intravesicular aqueous contents and lipid mixing of bilayers. The packing of the phospholipid molecules is greatly perturbed by the peptide, as deduced from the drastic changes induced by the peptide in cooperative properties associated with the phase transition of the bilayers. At saturating peptide/phospholipid ratios, the phase transition of dimyristoylphosphatidylglycerol vesicles is abolished. All of these effects are saturated at about 0.3 peptide/lipid molar ratio. The peptide adopts a mostly random structure in aqueous solution. A conformation composed of a high proportion of antiparallel beta-sheet is induced as a consequence of the interaction with the phospholipid vesicles in opposition to trifluoroethanol that promotes alpha-helical peptide structures, as deduced from circular dichroism measurements. The obtained results are discussed in terms of the potential involvement of the region comprising residues 116-139 of alpha-sarcin in the hydrophobic interactions of this cytotoxic protein with membranes.

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.

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles. The dilution of the fluorescence probes was faster when these were incorporated into the bilayers made of synthetic phospholipids than were present in those made of natural phospholipids. The bilayer destabilization produced by the protein in the vesices composed of the dimyristoyl-phospholipids should be transmitted to the more stable ones made of natural phospholipids. The obtained results are interpreted in terms of lipid mixing occurring within vesicle aggregates larger than dimer.

Comparative study on the secondary structure of lysozymes from different sources

The secondary structure of 16 type c lysozymes, 2 type g lysozymes, phage T4 lysozyme and bovine alpha-lactalbumin have been estimated according to the method of Chou and Fasman (1978). The prediction has been done on the partial sequence of lysozymes from three insects and tortoise egg-white lysozyme. From these studies, patterns of common secondary structure domains on lysozymes have been developed. These patterns have been compared attending to the active site and the antigenic loop of the hen enzyme of which three-dimensional structure is known. The three-dimensional structures of phage T4 and hen egg-white lysozymes have common alpha-carbon backbone domains being that also observed by these predictions. Immunological results are also discussed in terms of secondary structure homologies.

Implantation of sepiolite-collagen complexes in surgically created rat calvaria defects

The response of osseous tissue to the implantation of sepiolite-collagen complexes has been studied. Sepiolite, sepiolite-collagen complex and 0.5% glutaraldehyde-treated sepiolite-collagen complex were implanted in created circular defects in rat calvaria. The tissue reactions were analysed using light, transmission and scanning electron microscopies. The patterns of bone growth were radiographically analysed and the bone activity was indirectly quantified by using a point-count method. The reaction against the three implanted materials is characteristic of a foreign body reaction with abundant macrophages and giant cells. Implanted products have been detected in macrophages, which suggest the involvement of phagocytosis in the resorptive process. Bone grew at the implantation sites originating excrescences or sometimes a thin bridge at the defect margins. The studied materials, after implantation in contact with bone tissue, did not produce any toxic effect or necrosis, allowing bone activity.