Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein.

In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils' detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose. The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM). It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases. We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Partially Assembled Nucleosome Structures at Atomic Detail.

The evidence is now overwhelming that partially assembled nucleosome states (PANS) are as important as the canonical nucleosome structure for the understanding of how accessibility to genomic DNA is regulated in cells. We use a combination of molecular dynamics simulation and atomic force microscopy to deliver, in atomic detail, structural models of three key PANS: the hexasome (H2A·H2B)·(H3·H4)2, the tetrasome (H3·H4)2, and the disome (H3·H4). Despite fluctuations of the conformation of the free DNA in these structures, regions of protected DNA in close contact with the histone core remain stable, thus establishing the basis for the understanding of the role of PANS in DNA accessibility regulation. On average, the length of protected DNA in each structure is roughly 18 basepairs per histone protein. Atomistically detailed PANS are used to explain experimental observations; specifically, we discuss interpretation of atomic force microscopy, Förster resonance energy transfer, and small-angle x-ray scattering data obtained under conditions when PANS are expected to exist. Further, we suggest an alternative interpretation of a recent genome-wide study of DNA protection in active chromatin of fruit fly, leading to a conclusion that the three PANS are present in actively transcribing regions in a substantial amount. The presence of PANS may not only be a consequence, but also a prerequisite for fast transcription in vivo.

Matrix is everywhere: extracellular DNA is a link between biofilm and mineralization in Bacillus cereus planktonic lifestyle.

To date, the mechanisms of biomineralization induced by bacterial cells in the context of biofilm formation remain the subject of intensive studies. In this study, we analyzed the influence of the medium components on the induction of CaCO3 precipitation by the Bacillus cereus cells and composition of the extracellular matrix (ECM) formed in the submerged culture. While the accumulation of extracellular polysaccharides and amyloids appeared to be independent of the presence of calcium and urea during the growth, the accumulation of extracellular DNA (eDNA), as well as precipitation of calcium carbonate, required the presence of both ingredients in the medium. Removal of eDNA, which was sensitive to treatment by DNase, did not affect other matrix components but resulted in disruption of cell network formation and a sixfold decrease in the precipitate yield. An experiment with a cell-free system confirmed the acceleration of mineral formation after the addition of exogenous salmon sperm DNA. The observed pathway for the formation of CaCO3 minerals in B. cereus planktonic culture included a production of exopolysaccharides and negatively charged eDNA lattice promoting local Ca2+ supersaturation, which, together with an increase in the concentration of carbonate ions due to pH rise, resulted in the formation of an insoluble precipitate of calcium carbonate. Precipitation of amorphous CaCO3 on eDNA matrix was followed by crystal formation via the ACC-vaterite-calcite/aragonite pathway and further formation of larger mineral aggregates in complex with extracellular polymeric substances. Taken together, our data showed that DNA in extracellular matrix is an essential factor for triggering the biomineralization in B. cereus planktonic culture.

Antibacterial Properties of Fucoidans from the Brown Algae Fucus vesiculosus L. of the Barents Sea.

Fucoidans, sulfated polysaccharides found in cell walls of brown algae, are considered as a promising antimicrobial component for various applications in medicine and the food industry. In this study, we compare the antibacterial properties of two fractions of fucoidan from the brown algae Fucus vesiculosus gathered in the littoral of the Barents Sea and sampled at different stages of purification. The crude fraction of fucoidan was isolated from algae by extraction with aqueous ethanol and sonication. The purified fraction was obtained by additional treatment of the crude fraction with a solution of calcium chloride. The structural features of both fractions were characterized in detail and their antibacterial effects against several Gram-positive and Gram-negative bacteria were compared by photometry, acridine orange staining assay, and atomic force microscopy. Fucoidan inhibited growth in all of the above microorganisms, showing a bacteriostatic effect with minimum inhibitory concentrations (MIC) in the range between 4 and 6 mg/mL, with E. coli being the most sensitive to both fractions. Changes in the chemical composition after treatment of the crude fraction with a solution of calcium chloride led to a decrease in the content of sulfates and uronic acids and diminished antibacterial activity.

Transversal stiffness and Young's modulus of single fibers from rat soleus muscle probed by atomic force microscopy.

The structural integrity of striated muscle is determined by extra-sarcomere cytoskeleton that includes structures that connect the Z-disks and M-bands of a sarcomere to sarcomeres of neighbor myofibrils or to sarcolemma. Mechanical properties of these structures are not well characterized. The surface structure and transversal stiffness of single fibers from soleus muscle of the rat were studied with atomic force microscopy in liquid. We identified surface regions that correspond to projections of the Z-disks, M-bands, and structures between them. Transversal stiffness of the fibers was measured in each of these three regions. The stiffness was higher in the Z-disk regions, minimal between the Z-disks and the M-bands, and intermediate in the M-band regions. The stiffness increased twofold when relaxed fibers were maximally activated with calcium and threefold when they were transferred to rigor (ATP-free) solution. Transversal stiffness of fibers heavily treated with Triton X-100 was about twice higher than that of the permeabilized ones, however, its regional difference and the dependence on physiological state of the fiber remained the same. The data may be useful for understanding mechanics of muscle fibers when it is subjected to both axial and transversal strain and stress.

The amyloidogenicity of the influenza virus PB1-derived peptide sheds light on its antiviral activity.

The influenza virus polymerase complex is a promising target for new antiviral drug development. It is known that, within the influenza virus polymerase complex, the PB1 subunit region from the 1st to the 25th amino acid residues has to be is in an alpha-helical conformation for proper interaction with the PA subunit. We have previously shown that PB1(6-13) peptide at low concentrations is able to interact with the PB1 subunit N-terminal region in a peptide model which shows aggregate formation and antiviral activity in cell cultures. In this paper, it was shown that PB1(6-13) peptide is prone to form the amyloid-like fibrillar aggregates. The peptide homo-oligomerization kinetics were examined, and the affinity and characteristic interaction time of PB1(6-13) peptide monomers and the influenza virus polymerase complex PB1 subunit N-terminal region were evaluated by the SPR and TR-SAXS methods. Based on the data obtained, a hypothesis about the PB1(6-13) peptide mechanism of action was proposed: the peptide in its monomeric form is capable of altering the conformation of the PB1 subunit N-terminal region, causing a change from an alpha helix to a beta structure. This conformational change disrupts PB1 and PA subunit interaction and, by that mechanism, the peptide displays antiviral activity.

The dynamic wave expansion neural network model for robot motion planning in time-varying environments.

We introduce a new type of neural network--the dynamic wave expansion neural network (DWENN)--for path generation in a dynamic environment for both mobile robots and robotic manipulators. Our model is parameter-free, computationally efficient, and its complexity does not explicitly depend on the dimensionality of the configuration space. We give a review of existing neural networks for trajectory generation in a time-varying domain, which are compared to the presented model. We demonstrate several representative simulative comparisons as well as the results of long-run comparisons in a number of randomly-generated scenes, which reveal that the proposed model yields dominantly shorter paths, especially in highly-dynamic environments.

A conservative mutant of a proteolytic fragment produced during fibril formation enhances fibrillogenesis.

The fibrillogenesis of a peptide corresponding to residues 35-51 of human α-lactalbumin (¹GYDTQAIVENNESTEYG¹7) can be dramatically enhanced by the addition of a tetrapeptide TDYG homologous to its C-terminus (TEYG). Generation of spontaneous hydrolytic products similar to this peptide was demonstrated by mass-spectrometry analysis of GYDTQAIVENNESTEYG peptide solution components during fibrillogenesis. Possible mechanisms and roles of short peptides in protein metabolism are discussed.

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering.

Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations.

Amyloidogenic peptide homologous to fragment 129-148 of human myocilin.

Myocilin is a protein with a molecular weight near 50 kDa. It is expressed in almost all organs and tissues. We showed that the peptide DQLETQTRELETAYSNLLRD corresponding to N-terminal Leucine zipper motif (LZM) of the protein is able to form amyloid-like fibrils. The possible role of this motif in myocilin aggregation is discussed.

Structural Features of the Peptide Homologous to 6-25 Fragment of Influenza A PB1 Protein.

A mirror-symmetry motif was discovered in the N-terminus of the influenza virus PB1 protein. Structure of peptide comprised of the corresponding part of PB1 (amino acid residues 6-25) was investigated by circular dichroism and in silico modeling. We found that peptide PB1 (6-25) in solution assumes beta-hairpin conformation. A truncated peptide PB1 (6-13), containing only half of the mirror-symmetry motif, appeared to stabilize the beta-structure of the original peptide and, at high concentrations, was capable of reacting with peptide to form insoluble aggregates in vitro. Ability of PB1 (6-13) peptide to interact with the N-terminal domain of PB1 protein makes it a potential antiviral agent that inhibits PA-PB1 complex formation by affecting PB1 N-terminus structure.