Accumulation of storage proteins in plant seeds is mediated by amyloid formation.

Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved ß-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds.

Extent of N-Terminus Folding of Semenogelin 1 Cleavage Product Determines Tendency to Amyloid Formation.

It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86-107), SEM1(68-107), SEM1(49-107) and SEM1(45-107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45-107) and SEM1(49-107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45-107) starts immediately after purification, which is not observed for SEM1(49-107). Seeing that the peptide amino acid sequence of SEM1(45-107) differs from SEM1(49-107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45-67) and SEM1(49-67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45-67) and SEM1(49-67). However, SEM1(45-67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into ß-strands during amyloid formation process. Thus, the difference in full-length peptides' (SEM1(45-107) and SEM1(49-107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45-107) N-terminus, which contributes to an increased rate of amyloid formation.

Synthesis, biological evaluation and structure-activity relationship of 2-(2-hydroxyaryl)alkenylphosphonium salts with potency as anti-MRSA agents.

Here we report the synthesis, in vitro antimicrobial activity, preliminary toxicity and mechanism study of a new series of 2-(2-hydroxyaryl)alkenylphosphonium salts with the variation of phosphonium moiety obtained by a two-step synthetic method from phosphine oxides. The salts showed pronounced activity against Gram-positive bacteria, including MRSA strains, and some fungi. Mechanism of action against S. aureus was studied by CV test, TEM and proteomic assay. No cell wall integrity loss was observed while proteomic assay results suggested interference in different metabolic processes of S. aureus. For this series, lipophilicity was determined as a key factor for the inhibition of Gram-positive bacteria growth and S. aureus killing. Biological properties of methylated derivatives were notably different with manifested action against Gram-negative bacteria.

Thermodynamic vs. Kinetic Control in Synthesis of O-Donor 2,5-Substituted Furan and 3,5-Substituted Pyrazole from Heteropropargyl Precursor.

Elaboration of a convenient route towards donor-substituted pyrazoles from heteropropargyl precursors is challenging due to a number of thermodynamically favorable side reactions (e.g., acetylene-allene isomerization and Glaser homocoupling). In this work, Sonogashira cross-coupling conditions of 4-tert-butylphenyl propargyl ether with benzoyl chloride followed by tandem Michael addition/cyclocondensation with hydrazine into 3,5-disubstituted pyrazole (kinetic control), as well as cycloisomerization conditions of ketoacetylene intermediate into 2,5-disubstituted furan (thermodynamic control), were established through a variation of the catalyst loading, solvent polarity, excess of triethylamine, and time of reaction. During the optimization of process parameters, a number of by-products represented by a monophosphine binuclear complex (PPh3PdI2)2 with two bridging iodine atoms and diyne were identified and isolated in the pure form. The quantum-chemical calculations and solution-state 1H/13C NMR spectroscopy suggested that the 5(3)-(4-tert-butylphenyloxy)methoxy-3(5)-phenyl-1H-pyrazole exists in the tautomeric equilibrium in a polar methanol solvent and that individual tautomers could be characterized in case aprotic solvents employed. The pyrazole features a unique tetramer motif in the crystal phase formed by alternating 3(5)-phenyl-1H-pyrazole tautomers, which was stabilized by N-H···N bonds and stacking interactions of pyrazole rings, whereas pyrazole dimers were identified in the gas phase.

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization.

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Šresolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.

Solution structure of the N-terminal domain of the Staphylococcus aureus hibernation promoting factor.

Staphylococcus aureus hibernation promoting factor (SaHPF) is a 22,2 kDa protein which plays a crucial role in 100S Staphylococcus aureus ribosome formation during stress. SaHPF consists of N-terminal domain (NTD) that prevents proteins synthesis by binding to the 30S subunit at the P- and A-sites, connected through a flexible linker with a C-terminal domain (CTD) that keeps ribosomes in 100S form via homodimerization. Recently obtained 100S ribosome structure of S. aureus by cryo-EM shown that SaHPF-NTD bound to the ribosome active sites, however due to the absence of SaHPF-NTD structure it was modeled by homology with the E. coli hibernation factors HPF and YfiA. In present paper we have determined the solution structure of SaHPF-NTD by high-resolution NMR spectroscopy which allows us to increase structural knowledge about HPF structure from S. aureus.

Oligomerization of the antimicrobial peptide Protegrin-5 in a membrane-mimicking environment. Structural studies by high-resolution NMR spectroscopy.

Protegrin pore formation is believed to occur in a stepwise fashion that begins with a nonspecific peptide interaction with the negatively charged bacterial cell walls via hydrophobic and positively charged amphipathic surfaces. There are five known nature protegrins (PG1-PG5), and early studies of PG-1 (PDB ID:1PG1) shown that it could form antiparallel dimer in membrane mimicking environment which could be a first step for further oligomeric membrane pore formation. Later, we solved PG-2 (PDB ID:2MUH) and PG-3 (PDB ID:2MZ6) structures in the same environment and for PG-3 observed a strong dαα NOE effects between residues R18 and F12, V14, and V16. These "inconsistent" with monomer structure NOEs appears due to formation of an additional antiparallel ß-sheet between two monomers. It was also suggested that there is a possible association of protegrins dimers to form octameric or decameric ß-barrels in an oligomer state. In order to investigate a more detailed oligomerization process of protegrins, in the present article we report the monomer (PDB ID: 2NC7) and octamer pore structures of the protegrin-5 (PG-5) in the presence of DPC micelles studied by solution NMR spectroscopy. In contrast to PG-1, PG-2, and PG-3 studies, for PG-5 we observed not only dimer NOEs but also several additional NOEs between side chains, which allows us to calculate an octamer pore structure of PG-5 that was in good agreement with previous AFM and PMF data.

Use of a combination of the RDC method and NOESY NMR spectroscopy to determine the structure of Alzheimer's amyloid Aß10-35 peptide in solution and in SDS micelles.

The spatial structure of Alzheimer's amyloid Aß10-35-NH2 peptide in aqueous solution at pH 7.3 and in SDS micelles was investigated by use of a combination of the residual dipolar coupling method and two-dimensional NMR spectroscopy (TOCSY, NOESY). At pH 7.3 Aß10-35-NH2 adopts a compact random-coil conformation whereas in SDS micellar solutions two helical regions (residues 13-23 and 30-35) of Aß10-35-NH2 were observed. By use of experimental data, the structure of "peptide-micelle" complex was determined; it was found that Aß10-35-NH2 peptide binds to the micelle surface at two regions (residues 17-20 and 29-35).

Spatial structure of heptapeptide Aß(16-22) (beta-amyloid Aß(1-40) active fragment) in solution and in complex with a biological membrane model.

The spatial structure of an active fragment of beta-amyloid Aß(1-40) heptapeptide Aß(16-22) (Lys-Leu-Val-Phe-Phe-Ala-Glu) in aqueous buffer solution and in complex with sodium dodecyl sulfate micelles as a model membrane system was investigated by (1)H NMR spectroscopy and two-dimensional NMR (TOCSY, HSQC-HECADE (Heteronuclear Couplings from ASSCI-domain experiments with E.COSY-type crosspeaks), NOESY) spectroscopy. Complex formation was confirmed by the chemical shift changes of the heptapeptide's (1)H NMR spectra, as well as by the signs and values of the NOE effects in different environments. We compared the spatial structure of the heptapeptide in borate buffer solution and in complex with a model of the cell surface membrane.

Backbone and side chain NMR assignments for the ribosome maturation factor P (RimP) from Staphylococcus aureus.

The ribosomal maturation factor (RimP) is a 17.7 kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight ß-strands and three α-helices with the topology α1-ß1-ß2-α2- ß3- α3- ß4- ß5- ß6- ß7- ß8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.

Complexation of Oligo- and Polynucleotides with Methoxyphenyl-Functionalized Imidazolium Surfactants.

Interaction between cationic surfactants and nucleic acids attracts much attention due to the possibility of using such systems for gene delivery. Herein, the lipoplexes based on cationic surfactants with imidazolium head group bearing methoxyphenyl fragment (MPI-n, n = 10, 12, 14, 16) and nucleic acids (oligonucleotide and plasmid DNA) were explored. The complex formation was confirmed by dynamic/electrophoretic light scattering, transmission electron microscopy, fluorescence spectroscopy, circular dichroism, and gel electrophoresis. The nanosized lipoplex formation (of about 100-200 nm), contributed by electrostatic, hydrophobic interactions, and intercalation mechanism, has been shown. Significant effects of the hydrocarbon tail length of surfactant and the type of nucleic acid on their interaction was revealed. The cytotoxic effect and transfection ability of lipoplexes studied were determined using M-HeLa, A549 cancer cell lines, and normal Chang liver cells. A selective reduced cytotoxic effect of the complexes on M-HeLa cancer cells was established, as well as a high ability of the systems to be transfected into cancer cells. MPI-n/DNA complexes showed a pronounced transfection activity equal to the commercial preparation Lipofectamine 3000. Thus, it has been shown that MPI-n surfactants are effective agents for nucleic acid condensation and can be considered as potential non-viral vectors for gene delivery.

Trialkyl(vinyl)phosphonium Chlorophenol Derivatives as Potent Mitochondrial Uncouplers and Antibacterial Agents.

Trialkyl phosphonium derivatives of vinyl-substituted p-chlorophenol were synthesized here by a recently developed method of preparing quaternary phosphonium salts from phosphine oxides using Grignard reagents. All the derivatives with a number (n) of carbon atoms in phosphonium alkyl substituents varying from 4 to 7 showed pronounced uncoupling activity in isolated rat liver mitochondria at micromolar concentrations, with a tripentyl derivative being the most effective both in accelerating respiration and causing membrane potential collapse, as well as in provoking mitochondrial swelling in a potassium-acetate medium. Remarkably, the trialkyl phosphonium derivatives with n from 4 to 7 also proved to be rather potent antibacterial agents. Methylation of the chlorophenol hydroxyl group suppressed the effects of P555 and P444 on the respiration and membrane potential of mitochondria but not those of P666, thereby suggesting a mechanistic difference in the mitochondrial uncoupling by these derivatives, which was predominantly protonophoric (carrier-like) in the case of P555 and P444 but detergent-like with P666. The latter was confirmed by the carboxyfluorescein leakage assay on model liposomal membranes.

Towards Universal Stimuli-Responsive Drug Delivery Systems: Pillar[5]arenes Synthesis and Self-Assembly into Nanocontainers with Tetrazole Polymers.

In this work, we have proposed a novel universal stimulus-sensitive nanosized polymer system based on decasubstituted macrocyclic structures-pillar[5]arenes and tetrazole-containing polymers. Decasubstituted pillar[5]arenes containing a large, good leaving tosylate, and phthalimide groups were first synthesized and characterized. Pillar[5]arenes containing primary and tertiary amino groups, capable of interacting with tetrazole-containing polymers, were obtained with high yield by removing the tosylate and phthalimide protection. According to the fluorescence spectroscopy data, a dramatic fluorescence enhancement in the pillar[5]arene/fluorescein/polymer system was observed with decreasing pH from neutral (pH = 7) to acidic (pH = 5). This indicates the destruction of associates and the release of the dye at a pH close to 5. The presented results open a broad range of opportunities for the development of new universal stimulus-sensitive drug delivery systems containing macrocycles and nontoxic tetrazole-based polymers.

2D Monomolecular Nanosheets Based on Thiacalixarene Derivatives: Synthesis, Solid State Self-Assembly and Crystal Polymorphism.

Synthetic organic 2D materials are attracting careful attention of researchers due to their excellent functionality in various applications, including storage batteries, catalysis, thermoelectricity, advanced electronics, superconductors, optoelectronics, etc. In this work, thiacalix[4]arene derivatives functionalized by geranyl fragments at the lower rim in cone and 1,3-alternate conformations, that are capable of controlled self-assembly in a 2D nanostructures were synthesized. X-ray diffraction analysis showed the formation of 2D monomolecular-layer nanosheets from synthesized thiacalix[4]arenes, the distance between which depends on the stereoisomer used. It was established by DSC, FSC, and PXRD methods that the obtained macrocycles are capable of forming different crystalline polymorphs, moreover dimethyl sulphoxide (DMSO) is contributing to the formation of a more stable polymorph for cone stereoisomer. The obtained crystalline 2D materials based on synthesized thiacalix[4]arenes can find application in material science and medicine for the development of modern pharmaceuticals and new generation materials.

Facile Access to Optically Active 2,6-Dialkyl-1,5-Diazacyclooctanes.

The chiral substituted 1,5-diazacyclooctane (1,5-DACO) is of considerable importance and has attracted attention from a wide range of fields due to their unique chemical and biological properties. Despite the application potential, further study has not been optimized due to difficulties in their synthetic accessibility. Here, we report that the 1,5-DACO bearing a chiral auxiliary obtained from the formal [4+4] cycloaddition of N-alkyl-α,ß-unsaturated imines can be further derivatized by nucleophilic alkylation to give various chiral substituted 1,5-DACO derivatives. The removal of the chiral auxiliary was effectively carried out using hydrogenation over Pearlman's catalyst. This methodology allows the production of a broad range of unprecedented optically active 2,6-dialkyl-1,5-DACO, which could not be accessed by other methods.

Backbone and side chain NMR assignments for the ribosome binding factor A (RbfA) from Staphylococcus aureus.

Ribosome binding factor A (RbfA) is a 14.9 kDa adaptive protein of cold shock, which is important for bacterial growth at low temperatures. RbfA can bind to the free 30S ribosomal subunit and interacts with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is important for the efficient processing of 16S rRNA and for the maturation (assembly) of 30S ribosomal subunits. Here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of RbfA from Staphylococcus aureus. Analysis of the backbone chemical shifts by TALOS+ suggests that RbfA contains four α-helixes and three ß-strands with α1-ß1-ß2-α2-α3-ß3-α4 topology. Secondary structure of RbfA have KH-domain fold topology with ßααß subunit which is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, where an Ala residue at position 70 forming an interhelical kink. The solution of the structure of this protein factor and its complex with the ribosome by NMR spectroscopy, X-ray diffraction analysis and cryo-electron microscopy will allow further development of highly selective substances for slowing or completely stopping the translation of the pathogenic bacterium S. aureus, which will interfere with the synthesis and isolation of its pathogenicity factors.

NMR assignments of the N-terminal domain of Staphylococcus aureus hibernation promoting factor (SaHPF).

Staphylococcus aureus: hibernation-promoting factor (SaHPF) is a 22.2 kDa stationary-phase protein that binds to the ribosome and turns it to the inactive form favoring survival under stress. Sequence analysis has shown that this protein is combination of two homolog proteins obtained in Escherichia coli-ribosome hibernation promoting factor (HPF) (11,000 Da) and ribosome modulation factor RMF (6500 Da). Binding site of E. coli HPF on the ribosome have been shown by X-ray study of Thermus thermophilus ribosome complex. Hence, recent studies reported that the interface is markedly different between 100S from S. aureus and E. coli. Cryo-electron microscopy structure of 100S S. aureus ribosomes reveal that the SaHPF-NTD binds to the 30S subunit as observed for shorter variants of HPF in other species and the C-terminal domain (CTD) protrudes out of each ribosome in order to mediate dimerization. SaHPF-NTD binds to the small subunit similarly to its homologs EcHPF, EcYfiA, and a plastid-specific YfiA. Furthermore, upon binding to the small subunit, the SaHPF-NTD occludes several antibiotic binding sites at the A site (hygromycin B, tetracycline), P site (edeine) and E site (pactamycin, kasugamycin). In order to elucidate the structure, dynamics and function of SaHPF-NTD from S. aureus, here we report the backbone and side chain resonance assignments for SaHPF-NTD. Analysis of the backbone chemical shifts by TALOS+ suggests that SaHPF-NTD contains two α-helices and four ß-strands (ß1-α1-ß2-ß3-ß4-α2 topology). Investigating the long-term survival of S. aureus and other bacteria under antibiotic pressure could lead to advances in antibiotherapy.

Backbone and side chain NMR assignments for the ribosome Elongation Factor P (EF-P) from Staphylococcus aureus.

Elongation Factor P (EF-P) is a 20.5 kDa protein that provides specialized translation of special stalling amino acid motifs. Proteins with stalling motifs are often involved in various processes, including stress resistance and virulence. Thus it has been shown that the virulent properties of microorganisms can be significantly reduced if the work of EF-P is disrupted. In order to elucidate the structure, dynamics and function of EF-P from Staphylococcus aureus (S. aureus), here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of EF-P. Analysis of the backbone chemical shifts by TALOS+ suggests that EF-P contains 1 α-helix and 13 ß-strands (ß1-ß2-ß3-ß4-ß5-ß6-ß7-α1-ß8-ß9-ß10-ß11-ß12-ß13). The solution of the structure of this protein by NMR and X-ray diffraction analysis, as well as the structure of the ribosome complex by cryo-electron microscopy, will allow further screening of highly selective inhibitors of the translation of the pathogenic bacterium S. aureus. Here we report the almost complete 1H, 13C, 15N backbone and side chain NMR assignment of a 20.5 kDa EF-P.