Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncurium.

A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.     

Molecular dynamics study of a complex between the human histocompatibility antigen HLA-A2 and the IMP58-66 nonapeptide from influenza virus matrix protein.

The structure of the influenza-virus-matrix-protein (IMP) 58-66 nonapeptide, bound to the major-histocompatibility-complex-encoded human leukocyte antigen (HLA) A2 protein was studied by molecular dynamics simulation. Starting from the extra electron density map of peptides co-crystallized with HLA-A2, the nonapeptide IMP58-66 was docked residue by residue in the protein binding cleft. The complex was simulated for 100 ps in a shell of 1372 water molecules. The averaged simulated HLA-A2 conformation was found to be similar to the crystal structure (0.182 nm RMS deviation, for the backbone atoms of the alpha 1-alpha 2 domain). Nine out of the 14 hydrogen bonds observed in the antigen-binding site were reproduced in the simulation. The IMP58-66 peptide exhibits an extended conformation with kinks at positions 3 and 5. The side chains of residues 2, 3 and 9 develop van der Waals' interactions with hydrophobic pockets of HLA-A2, corresponding to polymorphic residues of the major-histocompatibility-complex-encoded proteins. Both the N-terminus and C-terminus of the nonapeptide were anchored in the antigen-binding groove by hydrogen bonds with conserved amino acids. The N-terminus was more flexible and contacts four HLA-A2 conserved tyrosines (Tyr7, Tyr59, Tyr159 and Tyr171) and Glu63 by direct or water-relayed hydrogen bonds. Water intercalation occurred only around the N-terminus of the peptide, the C-terminal carboxylate forming strong hydrogen bonds with polar residues (Tyr84 and Thr143) and a salt bridge with Lys146 all over the molecular dynamics simulation. This model is fully compatible with the recently published crystal structure of the HLA-B27 protein, complexed by a mixture of self nonapeptides.     

Radial Turgor and Osmotic Pressure Profiles in Intact and Excised Roots of Aster tripolium: Pressure Probe Measurements and Nuclear Magnetic Resonance-Imaging Analysis

High-resolution nuclear magnetic resonance images (using very short spin-echo times of 3.8 milliseconds) of cross-sections of excised roots of the halophyte Aster tripolium showed radial cell strands separated by air-filled spaces. Radial insertion of the pressure probe (along the cell strands) into roots of intact plants revealed a marked increase of the turgor pressure from the outermost to the sixth cortical layer (from about 0.1-0.6 megapascals). Corresponding measurements of intracellular osmotic pressure in individual cortical cells (by means of a nanoliter osmometer) showed an osmotic pressure gradient of equal magnitude to the turgor pressure. Neither gradient changed significantly when the plants were grown in, or exposed for 1 hour to, media of high salinity. Differences were recorded in the ability of salts and nonelectrolytes to penetrate the apoplast in the root. The reflection coefficients of the cortical cells were approximately 1 for all the solutes tested. Excision of the root from the stem resulted in a collapse of the turgor and osmotic pressure gradients. After about 15 to 30 minutes, the turgor pressure throughout the cortex attained an intermediate (quasistationary) level of about 0.3 megapascals. This value agreed well with the osmotic value deduced from plasmolysis experiments on excised root segments. These and other data provided conclusions about the driving forces for water and solute transport in the roots and about the function of the air-filled radial spaces in water transport. They also showed that excised roots may be artifactual systems.     

Amide isosteres of lexitropsins: synthesis, DNA binding characteristics and sequence selectivity of thioformyldistamycin.

The synthesis and properties of an amide isostere of the antibiotic distamycin, thioformyldistamycin 3 is described. Compound 3 exists predominantly in the E conformation of the thioamide group in freshly prepared DMSO solution but is converted into the Z form, predicted by molecular mechanics to be more stable, on standing for 24 h. The coalescence temperature in DMSO is 110 degrees C by 1H-NMR. The thioformyl moiety of 3 is resistant to both peptidase action and acid treatment. Complementary strand MPE footprinting on a EcoRI/Hind III restriction fragment of pBR322 DNA demonstrated that either E or Z forms of 3 give a single set of footprints very similar to that of the parent antibiotic with strongest protection at TAAG and TATTAT with moderately strong protection at ATTT and AAAA. The strength of binding of 3 and distamycin from delta Tm measurements to either poly.d(AT) or calf thymus DNA is comparable. Molecular modeling predicted a preferred conformation for 3 wherein the C = S bond has a torsional angle of 110 degrees with the pyrrole ring. The energy difference between this conformation and the E form is less than 1 kcal/mole. In contrast the E-form has an energy 17.3 kcal/mole greater than the Z and a value of 26.3 kcal/mole was calculated for the energy barrier between the two isomers.     

γ-Glutamylcysteinylserine — A New Homologue of Glutathione in Plants of the Family Poaceae*

In addition to glutathione (γ-GluCysGly), many species of the family Poaceae have another tripeptide which has the amino acid sequence γ-GluCysSer. This thiol was isolated from etiolated leaves of wheat (Triticum aestivum L. cv. Star). Its structure was elucidated by quantitative amino acid analysis after total hydrolysis and by partial hydrolysis with carboxypeptidase A and γ-glutamyltranspeptidase. The content of γ-GluCysSer in the leaves of T. aestivum is increased by incubation with sulfate and is severely diminished by incubation with buthionine sulfoximine, a specific inhibitor of γ-glutamylcysteine synthetase. Oxidized γ-GluCysSer is reduced by yeast glutathione reductase with a rate somewhat lower than for glutathione, but the new tripeptide is not a substrate of glutathione-S-transferase from equine liver. Besides homoglutathione (γ-GluCysßAla), a tripeptide found in plants of the order Fabales, the tripeptide γ-GluCysSer is the second homologue of glutathione detected in plants.     

The S3 state of photosystem II: differences between the structure of the manganese complex in the S2 and S3 states determined by X-ray absorption spectroscopy

O2-evolving photosystem II (PSII) membranes from spinach have been cryogenically stabilized in the S3 state of the oxygen-evolving complex. The cryogenic trapping of the S3 state was achieved using a double-turnover illumination of dark-adapted PSII preparations maintained at 240 K. A double turnover of PSII was accomplished using the high-potential acceptor, Q400, which is the high-spin iron of the iron-quinone acceptor complex. EPR spectroscopy was the principal tool establishing the S-state composition and defining the electron-transfer events associated with a double turnover of PSII. The inflection point energy of the Mn X-ray absorption K-edge of PSII preparations poised in the S3 state is the same as for those poised in the S2 state. This is surprising in light of the loss of the multiline EPR signal upon advancing to the S3 state. This indicates that the oxidative equivalent stored within the oxygen-evolving complex (OEC) during this transition resides on another intermediate donor which must be very close to the manganese complex. An analysis of the Mn extended X-ray absorption fine structure (EXAFS) of PSII preparations poised in the S2 and S3 states indicates that a small structural rearrangement occurs during this photoinduced transition. A detailed comparison of the Mn EXAFS of these two S states with the EXAFS of four multinuclear mu-oxo-bridged manganese compounds indicates that the photosynthetic manganese site most probably consists of a pair of binuclear di-mu-oxo-bridged manganese structures. However, we cannot rule out, on the basis of the EXAFS analysis alone, a complex containing a mononuclear center and a linear trinuclear complex. The subtle differences observed between the S states are best explained by an increase in the spread of Mn-Mn distances occurring during the S2----S3 state transition. This increased disorder in the manganese distances suggests the presence of two inequivalent di-mu-oxo-bridged binuclear structures in the S3 state.     

Proton NMR studies of transforming and nontransforming H-ras p21 mutants

One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the Hl' proton and the alpha-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP.Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP.Mg2+.     

Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm

Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.     

Estrogen-dependent changes in the functional interrelationships among neurons,ependymal cells and glial cells of the arcuate nucleus

Summary The synchronizing effect of ethinylestradiol (4 g/g b.w.) on neurons of the arcuate nucleus 700–950 m caudal to the posterior edge of the optic chiasma was studied by karyometry in 6-week-old albino mice during proestrus.The caudal portion of the arcuate nucleus was identified as the most estrogen-sensitive subdivision; all neurons showed an increase in their nuclear area (mean transect, profile area of the nucleus) 1 h following administration of ethinylestradiol. This hypothalamic region was selected for the subsequent electron-microscopic cytometric study to analyze functional interrelationships among neurons, ependymal cells and glial cells. Six and 12 days after ovariectomy no significant change in the nuclear area of neurons and ependymal cells was found 850–950 m behind the posterior slope of the optic chiasma, but the neurons exhibited a decrease in the number of polyribosomes, the volume fraction (V_Vmi) and the surface density of the inner membrane of mitochondria (S_Vmi). A similar decrease in V_Vmi and S_Vmi was measured in the apical part of ependymal cells and in the pericapillary profiles of ependymal and glial cells, which was accompanied by a reduction in the surface density of ependymal processes extending into the ventricular lumen. In addition, no change of V_Vmi and S_Vmi was seen in the basal subnuclear part of ependymal cells.This bipolar functional reaction of ependymal cells after ovariectomy is discussed as an indicator of ependymal control of neuronal activity by sequestering biologically active agents, e.g., transmitters of neurohormones, in their apical and basal extensions facing the ventricular surface or the pericapillary space.