Yuriy F. Zuev

Chaperone-like activities of different molecular forms of β-casein. Importance of polarity of N-terminal hydrophilic domain

As a member of intrinsically unstructured protein family, β‐casein (β‐CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone‐like activity in vitro. Like many chaperones, native β‐CN does not contain cysteinyl residues and exhibits strong tendencies for self‐association. The chaperone‐like activities of three recombinant β‐CNs wild type (WT) β‐CN, C4 β‐CN (with cysteinyl residue in position 4) and C208 β‐CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native β‐CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β‐CND) of C4‐β‐CN and C208 β‐CN were also studied and their chaperone‐like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β‐CN, C208 β‐CN, C4 β‐CN and C4 β‐CND exhibited significantly lower chaperone‐like activities than native β‐CN. Dimerization of C208 β‐CN with two distal hydrophilic domains considerably improved its chaperone‐like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N‐terminal hydrophilic domain as important functional elements in enhancing the chaperone‐like activity of native β‐CN.

Circulating Microparticles Alter Formation, Structure, and Properties of Fibrin Clots

Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure, and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure, and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1–0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure, and resistance to fibrinolysis.

Physicochemical properties of complex rhamnogalacturonan I from gelatinous cell walls of flax fibers.

The physicochemical properties of flax fiber cell wall rhamnogalacturonan I (RG-I) and its fragments, obtained after galactanase treatment (fraction G1), were characterized. RG-I retains its hydrodynamic volume after its molecular weight decreases by approximately half, as revealed by SEC. Two techniques, DLS and NMR, with different principles of diffusion experiment were used to establish the reasons for this property of RG-I. Three possible types of particles were revealed by DLS depending on the concentration of the RG-I and G1 solutions (2-2.5, 15-20, and 150-200 nm). It was determined by BPP-LED experiments that the backbone of the RG-I was 1.3-1.9-fold more mobile than the side chains. The obtained data suggest a novel type of pectin spatial organization-the formation of RG-I associates with the backbone at the periphery and the interaction between the side chains to form a core zone.

Structural, biocomplexation and gene delivery properties of hydroxyethylated gemini surfactants with varied spacer length.

Gemini surfactants with hexadecyl tails and hydroxyethylated head groups bridged with tetramethylene (G4), hexamethylene (G6) and dodecamethylene (G12) spacers were shown to self-assemble at the lower critical micelle concentration compared to their conventional m-s-m analogs. The lipoplex formation and the plasmid DNA transfer into different kinds of host cells were studied. In the case of eukaryotic cells, high transfection efficacy has been demonstrated for DNA-gemini complexes, which increased as follows: G6<G4<G12. Different activity series, i.e., G6>G4>G12 has been obtained in the case of transformation of bacterial cells with plasmid DNA-gemini complexes, mediated by electroporation technique. Solely G6 shows transformation efficacy exceeding the control result (uncomplexed DNA), while the inhibitory effect occurs for G4 and G12. Analysis of physico-chemical features of single surfactants and lipoplexes shows that compaction and condensation effects change as follows: G6<G4 ≤ G12, i.e., agree with the order of transfection efficacy, which is supported by membrane tropic properties of G12. On the other hand, gel retardation assay and docking study testify low electrostatic affinity in G12/DNA pair, thereby indicating that hydrophobic effect probably plays important role in the lipoplex formation. Two factors are assumed to be responsible for the inhibition effect of gemini in the case of transformation of bacterial cells. They are (i) an unfavorable influence of cationic surfactants on the electroporation procedure due to depressing the electrophoretic effect; and (ii) antibacterial activity of cationic surfactants that may cause the disruption of integrity of cell membranes.

Novel biomimetic systems based on amphiphilic compounds with a diterpenoid fragment: Role of counterions in self-assembly

Novel biomimetic systems are designed based on cationic surfactants composed of an isosteviol moiety and different counterions, namely bromide (S1) and tosylate (S2). The counterion structure is shown to play a crucial role in the surfactant association. A number of methods used provide evidence that only one type of aggregate, i.e., micelles are observed in the S2 systems, while a concentration-dependent association occurs in the case of S1. The DLS and fluorescence anisotropy measurements reveal that the micelle-vesicle-micelle transitions probably occur with the S1 system. The occurrence of small aggregates near the critical micelle concentration with radii of 2.5 nm is supported by NMR self-diffusion data. The Orange OT solubilization results strongly support the idea of a second threshold in the S1 system around 0.025 mM and provide evidence that hydrophobic domains occur in the aggregates. The latter property and the capacity to integrate with the lipid bilayer make it possible to suggest the newly synthesized surfactants as effective nanocontainers for hydrophobic guests.

Response of pigmented Serratia marcescens to the illumination.

Variations in the illumination conditions (light/darkness) affected both the biosynthesis of prodigiosin and energy metabolism of the pigmented strain ATCC 9986 Serratia marcescens growing aerobical in the batch culture were shown. In the process incubation the transition of the pigmented culture from illumination within (24 h, 48 h) in the dark conditions increased the prodigiosin synthesis by 2.0, 2.5 times, respectively. At the same time, the illumination did not influence the prodigiosin biosynthesis in the stationary growth phase. In the initial period of prodigiosin synthesis the rate of oxygen consumption was higher than later when the pigment synthesis gradually decreased. The respiration activity of colorless strain 24-5 is not independent from the lighting conditions. The regulation of energetic pathways in the light and in darkness has been revealed. Prodigiosin is associated with the hydrophobic protein and it is represented pigment protein complex by diameter of particles less 100 kDa. Fluorescence spectrum of prodigiosin and it the absorption spectra of derivatives of high orders D(IV) and D(VIII) were described.

Metrics of rhamnogalacturonan I with β-(1→4)-linked galactan side chains and structural basis for its self-aggregation

Within the family of plant cell wall polysaccharides rhamnogalacturonans I are the most diverse and structurally complex members. In present study we characterize the 3-dimensional structures and dynamic features of the constituents of RG-I along MD trajectories. It is demonstrated that extended threefold helical structure of the rhamnogalacturonan linear backbone is the most energetically favorable motif. Branching helps to stabilize a conformer of the backbone twisted along 1→2 glycosidic linkage triggering the orientation of long side chains without altering the extended overall backbone chain conformation. Formation of anti-parallel pairing of the β-galactan side chains allows us to suggest a novel mode of non-covalent cross-linking in pectins. Studied structural elements are organized to report the first attempt to characterize 3D structure of RG-I focusing on the special case of flax tertiary cell wall and elucidate the structural basis underlying the formation of RG-I self-associates and functional role of RG-I in planta.

Dynamics and thermodynamic properties of CXCL7 chemokine

Chemokines form a family of signaling proteins mainly responsible for directing the traffic of leukocytes, where their biological activity can be modulated by their oligomerization state. We characterize the dynamics and thermodynamic stability of monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines, using experimental methods that include circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, and computational methods that include the anisotropic network model (ANM), molecular dynamics (MD) simulations and the distance constraint model (DCM). A consistent picture emerges for the effects of dimerization and Cys5‐Cys31 and Cys7‐Cys47 disulfide bonds formation. The presence of disulfide bonds is not critical for maintaining structural stability in the monomer or dimer, but the monomer is destabilized more than the dimer upon removal of disulfide bonds. Disulfide bonds play a key role in shaping the characteristics of native state dynamics. The combined analysis shows that upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present, and the homodimer is least stable relative to its two monomers. These results suggest that the highly conserved disulfide bonds in chemokines facilitate a structural mechanism that is tuned to optimally distinguish functional characteristics between monomer and dimer. Proteins 2015; 83:1987–2007.

Conformational Flexibility and Self-Association of Fibrinogen in Concentrated Solutions

We studied the hydrodynamic behavior of fibrinogen, a blood plasma protein involved in blood clotting, in a broad 0.3-60 mg/mL range of concentration and 5-42 °C temperature using pulsed-field gradient 1H NMR-diffusometry. Arrhenius plots revealed the activation energy for fibrinogen diffusion Ed = 21.3 kJ/mol at 1.4 mg/mL and 28.4 kJ/mol at 38 mg/mL. We found a dramatic slowdown in fibrinogen self-diffusion with concentration beginning at 1.7-3.4 mg/mL, which deviated from the standard hard-particle behavior, suggesting a remarkable intermolecular entanglement. This concentration dependence was observed regardless of the absence or presence of the GPRP peptide (inhibitor of fibrin polymerization), and also in samples free of fibrin oligomers. By contrast, diffusivity of fibrinogen variant I-9 with truncated C-terminal portions of the Aα chains was much less concentration-dependent, indicating the importance of intermolecular linkages formed by the αC regions. Theoretical models combined with all-atom molecular dynamics simulations revealed partially bent fibrinogen solution conformations that interpolate between a flexible chain and a rigid rod observed in the crystal. The results obtained illuminate the important role of the αC regions in modulating the fibrinogen molecular shape through formation of weak intermolecular linkages that control the bulk properties of fibrinogen solutions.

Structure of Scots pine defensin 1 by spectroscopic methods and computational modeling.

Defensins are part of the innate immune system in plants with activity against a broad range of pathogens, including bacteria, fungi and viruses. Several defensins from conifers, including Scots pine defensin 1 (Pinus sylvestris defensin 1, (PsDef1)) have shown a strong antifungal activity, however structural and physico-chemical properties of the family, needed for establishing the structure-dynamics-function relationships, remain poorly characterized. We use several spectroscopic and computational methods to characterize the structure, dynamics, and oligomeric state of PsDef1. The three-dimensional structure was modeled by comparative modeling using several programs (Geno3D, SWISS-MODEL, I-TASSER, Phyre(2), and FUGUE) and verified by circular dichroism (CD) and infrared (FTIR) spectroscopy. Furthermore, FTIR data indicates that the structure of PsDef1 is highly resistant to high temperatures. NMR diffusion experiments show that defensin exists in solution in the equilibrium between monomers and dimers. Four types of dimers were constructed using the HADDOCK program and compared to the known dimer structures of other plant defensins. Gaussian network model was used to characterize the internal dynamics of PsDef1 in monomer and dimer states. PsDef1 is a typical representative of P. sylvestris defensins and hence the results of this study are applicable to other members of the family.