Justyna Czyrko

Triple helical collagen-like peptide interactions with selected polyphenolic compounds.

Because collagen is the most abundant component of connective tissue, it is an excellent biomaterial in numerous medical applications. However, the utility of collagen is limited by its low mechanical strength in aqueous solutions and its susceptibility to proteolytic degradation in vivo. To improve the physical properties of collagen and to enhance its chemical resistance, it is necessary to stabilize its structure through chemical or physical modifications. In this study, we analyzed the interactions of a model molecule, a synthetic triple helical collagen-like peptide, with polyphenols such as curcumin, rutin, quercetin, naringin, and hypericin. Interactions between the peptide and polyphenolic compounds were analyzed using various techniques. The layer-by-layer assembly processes of a gold surface using the peptide and polyphenols was performed via surface plasmon resonance (SPR), atomic force microscopy (AFM), and ellipsometry. SPR screening of polyphenols was conducted in real time to select compounds that bind to the collagen-like peptide and could thus be applied to the stabilization of collagen. Selected polyphenols, especially naringin and hypericin, demonstrated notable binding to the peptide. To determine the nature of these interactions, experiments were supplemented with crystallographic studies and molecular docking of plant metabolites and collagen-like peptides.

S-adenosyl-L-homocysteine hydrolase from a hyperthermophile (Thermotoga maritima) is expressed in Escherichia coli in inactive form – Biochemical and structural studies

Thermotoga maritima is a hyperthermophilic bacterium but its genome encodes a number of archaeal proteins including S-adenosyl-L-homocysteine hydrolase (SAHase), which regulates cellular methylation reactions. The question of proper folding and activity of proteins of extremophilic origin is an intriguing problem. When expressed in E.coli and purified (as a homotetramer) at room temperature, the hyperthermophilic SAHase from T.maritima was inactive. ITC study indicated that the protein undergoes heat-induced conformational changes, and enzymatic activity assays demonstrated that these changes are required to attain enzymatic activity. To explain the mechanism of thermal activation, two crystal structures of the inactive form of T. maritima SAHase (iTmSAHase) were determined for an incomplete binary complex with the reduced cofactor (NADH), and in a mixture of binary complexes with NADH and with adenosine. In contrast to active SAHases, in iTmSAHase only two of the four subunits contain a bound cofactor, predominantly in its non-reactive, reduced state. Moreover, the closed-like conformation of the cofactor-containing subunits precludes substrate delivery to the active site. The two other subunits cannot be involved in the enzymatic reaction either; although they have an open-like conformation, they do not contain the cofactor, whose binding site may be occupied by an adenosine molecule. The results suggest that this enzyme, when expressed in mesophilic cells, is arrested in the activity-incompatible conformation revealed by its crystal structures.

Carbon nano-onion composites: Physicochemical characteristics and biological activity

ABSTRACT Carbon nano-onion/surfactant (CNO/surfactant) composites offer the possibility to easily produce the soluble nanostructures. That approach combines the hydrophilicity of surfactants with the robustness of carbon structures to produce composites with superior and unusual physicochemical properties. We used the following surfactants: hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100), and polyethylene glycol sorbitan monolaurate (Tween 20) to non-covalently modify CNO surfaces. The existence of stable CNO composites are clearly evidenced by direct transmission electron microscopy observations, which are also supported by thermogravimetric analyses. Dynamic light scattering and zeta potential confirmed their dispersion and stability. Additionally, the biological activity of well-dispersed CNO/surfactant composites against a strain of Escherichia coli was assayed. In vitro antimicrobial assays for the composites revealed that only the CNO/CTAB composite decreased cell viability. This activity could be assigned to the simple composite dissociation in water solutions, however antimicrobial properties of the composite are slightly better when compared with pure CTAB. This indicate some synergic effect with respect to the properties of the pure surfactant.

Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa.

S-adenosyl-l-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K+ ion and one Zn2+ ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K+ ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn2+ ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K+ or Rb+ ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a 23Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn2+ ion is bound in the area of a molecular gate that regulates access to the active site. Zn2+ coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.

Crystal Structure of S-adenosyl-L-homocysteine Hydrolase from Cytophaga hutchinsonii, a Case of Combination of Crystallographic and Non-crystallographic Symmetry

The majority of living organisms utilize S-adenosyl-L-homocysteine hydrolase (SAHase) as a key regulator of cellular methylation reactions. The unusual evolution history of SAHase genes is reflected in the phylogeny of these proteins, which are grouped into two major domains: mainly archaeal and eukaryotic/bacterial. Such a phylogeny is in contradiction to the three-domain topology of the tree of life, commonly based on 16S rRNA sequences. Within the latter domain, SAHases are classified as eukaryotic-only or bacterial-only clades depending on their origin and sequence peculiarities. A rare exception in this classification is SAHase from a cellulose-utilizing soil bacterium Cytophaga hutchinsonii (ChSAHase), as the phylogenetic analyses indicate that ChSAHase belongs to the animal clade. Here, the P21212 crystal structure of recombinant ChSAHase in ternary complex with the oxidized form of the NAD+ cofactor and a reaction product/substrate (adenosine) is presented. Additionally, a sodium cation was identified in close proximity of the active site. The crystal contains two translational NCS-related intimate dimers of ChSAHase subunits in the asymmetric unit. Two complete tetrameric enzyme molecules are generated from these dimers within the crystal lattice through the operation of crystallographic twofold axes in the z direction.

Tetra­ethyl­ammonium toluene-4-sulfon­ate

There are two tetraethylammonium cations and two toluene-4-sulfate anions in the asymmetric unit of the title salt, C8H20N+·C7H7O3S−. One of the anions is disordered over two positions, with refined occupancies of 0.447 (3) and 0.553 (3). In the crystal, the cations and anions are linked by C—H⋯O hydrogen bonds, forming ribbons along [10-1]. The ribbons are linked via C—H⋯O hydrogen bonds, forming a two-dimensional network lying parallel to (10-1).

Tris(1,10-phenanthroline-κ2N,N′)ruthenium(II) bis­(perchlorate)

The asymmetric unit of the title compound, [Ru(C12H8N2)3](ClO4)2, contains one octahedrally coordinated RuII cation of the ruthenium-phenanthroline complex and three differently occupied perchlorate anions: two, denoted A and B, are located on the twofold axis while another, denoted C, is positioned in the proximity of the twofold screw axis. Perchlorate anions B and C are severely disordered. The occupancies of the two major conformers of anion B refined to 0.302 (6) and 0.198 (6). Perchlorate ion C was modeled in two alternate conformations which refined to occupancies of 0.552 (10) and 0.448 (10).