Furoquinoline alkaloids from the southern African Rutaceae Teclea natalensis

The chloroform and ethyl acetate extracts of the leaves of Teclea natalensis have yielded two furoquinoline alkaloids, 6-[(2,3-epoxy-3-methylbutyl)oxy]-4,7-dimethoxyfuro[2,3-b]quinoline and 4,7-dimethoxy-6-[(3-methyl-2-butenyl)oxy]furo[2,3-b]quinoline, and the known alkaloids 4,7-dimethoxy-8-[(3-methyl-2-butenyl)oxy]furo[2,3-b]quinoline, flindersiamine and dictamnine.     

Oligomerization paths of the nucleoprotein of influenza A virus

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.     

Structures and free-energy landscapes of the wild type and mutants of the Abeta(21-30) peptide are determined by an interplay between intrapeptide electrostatic and hydrophobic interactions

The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Aβ_21-30 peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated pK_a values that compare well with the experimental estimates. The pK_a values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22-K28 are more probable in “dried” salt bridges, whereas D23-K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23N < WT < E22Q < K28A. Free-energy profiles and disconnectivity representation of the energy landscapes show that the monomer structures partition into four distinct basins. The hydrophobic interactions cluster the Aβ_21-30 peptide into two basins, differentiated by the relative position of the DVG(23-25) and GSN(25-27) fragments about the G25 residue. The E22Q mutation increases the population with intact VGSN turn compared to the wild-type (WT) peptide. The increase in the population of the structures in the aggregation-prone Basin I in E22Q, which occurs solely due to the difference in charge states between the Dutch mutant and the WT, gives a structural explanation of the somewhat larger aggregation rate in the mutant. The D23N mutation dramatically reduces the intrapeptide interactions. The K28A mutation increases the intrapeptide hydrophobic interactions that promote population of structures in Basin I and Basin II whose structures are characterized by hydrophobic interaction between V24 and K28 side chains but with well-separated ends of the backbone atoms in the VGSN turn. The intrapeptide electrostatic interactions in the WT and E22Q peptides roughen the free-energy surface compared to the K28A peptide. The D23N mutation has a flat free-energy surface, corresponding to an increased population of random coil-like structures with weak hydrophobic and electrostatic interactions. We propose that mutations or sequences that enhance the probability of occupying Basin I would promote aggregation of Aβ peptides.     

Selective probing of a NADPH site controlled light‐induced enzymatic catalysis

Achieving molecular recognition of NADPH binding sites is a compelling strategy to control many redox biological processes. The NADPH sites recognize the ubiquitous NADPH cofactor via highly conserved binding interactions, despite differences in the regulation of the hydride transfer in redox active proteins. We recently developed a photoactive NADPH substitute, called nanotrigger NT synchronizing the initiation of enzymatic catalysis of the endothelial NO‐synthase (eNOS) with a laser pulse. Spatial and temporal control of enzymatic activity by such a designed light‐driven activator would benefit from achieving molecular selectivity, i.e. activation of a single NADPH‐mediated enzyme. In this work, we probe the ability of NT to discriminate between two NADPH sites with light. The selected NADPH sites belong to dihydrofolate reductase dihydrofolate reductase enzyme (DHFR) and endothelial NO‐synthase (eNOS). Ultrafast kinetics showed that NT could not activate DHFR catalysis with a laser pulse in contrast with the observed trigger of eNOS catalysis leading to NO formation. Homology modelling, molecular dynamics simulations showed that NT discriminated between the two NADPH sites by different donor to acceptor distances and by local steric effects hindering light activation of DHFR catalysis. The data suggested that the narrow NADPH site required a tight fit of the nanotrigger at a suitable distance/angle to the electron acceptor for a specific activation of the catalysis. The ability of the nanotrigger to activate eNOS combined with a low reactivity in unfavourable NADPH sites makes NT a highly promising tool for targeting eNOS in endothelial cells with a laser pulse. Copyright © 2009 John Wiley & Sons, Ltd.     

Probing the initial stage of aggregation of the Abeta(10-35)-protein: assessing the propensity for peptide dimerization

Characterization of the early stages of peptide aggregation is of fundamental importance in elucidating the mechanism of the formation of deposits associated with amyloid disease. The initial step in the pathway of aggregation of the Abeta-protein, whose monomeric NMR structure is known, was studied through the simulation of the structure and stability of the peptide dimer in aqueous solution. A protocol based on shape complementarity was used to generate an assortment of possible dimer structures. The structures generated based on shape complementarity were evaluated using rapidly computed estimates of the desolvation and electrostatic interaction energies to identify a putative stable dimer structure. The potential of mean force associated with the dimerization of the peptides in aqueous solution was computed for both the hydrophobic and the electrostatic driven forces using umbrella sampling and classical molecular dynamics simulation at constant temperature and pressure with explicit solvent and periodic boundary conditions. The comparison of the two free energy profiles suggests that the structure of the peptide dimer is determined by the favorable desolvation of the hydrophobic residues at the interface. Molecular dynamics trajectories originating from two putative dimer structures indicate that the peptide dimer is stabilized primarily through hydrophobic interactions, while the conformations of the peptide monomers undergo substantial structural reorganization in the dimerization process. The finding that the phi-dimer may constitute the ensemble of stable Abeta(10-35) dimer has important implications for fibril formation. In particular, the expulsion of water molecules at the interface might be a key event, just as in the oligomerization of Abeta(16-22) fragments. We conjecture that events prior to the nucleation process themselves might involve crossing free energy barriers which depend on the peptide-peptide and peptide-water interactions. Consistent with existing experimental studies, the peptides within the ensemble of aggregated states show no signs of formation of secondary structure.     

Flavonoids from Tephrosia aequilata

From the roots of the plant Tephrosia aequilata Baker, five flavonoids were isolated of which, 3,4:8,9-dimethylenedioxypterocarpan is reported for the first time. Its structure and those of the already known flavonoids were established by physical and spectroscopic analysis. Application of 2D NMR techniques was useful for complete characterization of the new pterocarpan as well as the other known flavonoids.