Marta Bruix

Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins.

The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI = 3.5) and a basic variant (pI = 10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulombs Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulombs Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.

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.

The Interaction of Hevein with N-acetylglucosamine-containing Oligosaccharides. Solution Structure of Hevein Complexed to Chitobiose

The three-dimensional structure of hevein, a small protein isolated from the latex of Hevea brasiliensis (rubber tree), in water solution has been obtained by using 1H-NMR spectroscopy and dynamic simulated annealing calculations. The average root-mean-square deviation (rmsd) of the best 20 refined structures generated using DIANA prior to simulated annealing was 0.092 nm for the backbone atoms and 0.163 nm for all heavy atoms (residues 3-41). The specific interaction of hevein with N-acetylglucosamine-containing oligosaccharides has also been analyzed by 1H-NMR. The association constants, Ka, for the binding of hevein to GlcNAc, chitobiose [GlcNAc-beta(1-->4)-GlcNAc], chitotriose [GlcNAc-beta(1-->4)-GlcNAc-beta(1-->4)-GlcNAc], and GlcNAc-alpha(1-->6)-Man have been estimated from 1H-NMR titration experiments. Since the measured Ka values for chitobiose binding are almost identical with and without calcium ions, it is shown that these cations are not required for sugar binding. The association increases in the order GlcNAc-alpha(1-->6)-Man 6)-Man can be explained by favourable stacking of the second beta-linked GlcNAc moiety and Trp21.

Aromatic polymers obtained by precipitation polycondensation: 4. Synthesis of poly(ether ketone ketone)s☆

Abstract High molecular weight aromatic poly(ether ketone ketone)s were synthesized by the Friedel-Crafts polyacylation condensation of iso- and terephthaloyl chlorides with diphenyl ether, 1,4- and 1,3-bis(4-phenoxybenzoyl)benzenes. Depending on the monomers used for polycondensation, polyketones of regular structure with different iso-/tereisomer repeating unit ratio ( 100 0 , 50 50 , 0 100 ) in the main chain were obtained. Polymers of each repeating isomer unit were prepared in two different ways. All the polymer syntheses were performed as precipitation polycondensations and the resulting polymers were obtained in particle form. The influence of reaction conditions and preparation route on the polymer properties were examined. The monomer concentration and monomer stoichiometric ratio were found to affect the polymer viscosity. The size and shape of the polyketone particles obtained were also found to be governed by reaction conditions and preparation route.

Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter sulfurreducens Reveals Mechanistic and Functional Diversity

A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c(7) from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e(-)/H(+) transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e(-)/H(+) coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G. sulfurreducens.

Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes

The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III-I-IV for PpcB, as opposed to I-IV-III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.

The behavior of sea anemone actinoporins at the water-membrane interface.

Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.

NMR and SAXS characterization of the denatured state of the chemotactic protein Che Y: Implications for protein folding initiation

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X‐ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that ∼25% of the residues are involved in residual structures. Conformational shifts of the α‐protons, heteronuclear 15N‐{1H} NOEs and 15N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1–70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone 1H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state.

Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin

α‐Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis—histidine‐50, glutamic acid‐96, and histidine‐137—were changed to glutamine. Three dif‐ ferent single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three‐dimensional structure of these variants of α‐sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of α‐sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild‐type α‐sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α‐sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α‐sarcin. Proteins 1999;37:474–484. ©1999 Wiley‐Liss, Inc.