Hierarchical clustering of the correlation patterns: new method of domain identification in proteins.

New method of identification of dynamical domains in proteins - Hierarchical Clustering of the Correlation Patterns (HCCP) is proposed. HCCP allows to identify the domains using single three-dimensional structure of the studied proteins and does not require any adjustable parameters that can influence the results. The method is based on hierarchical clustering performed on the matrices of correlation patterns, which are obtained by the transformation of ordinary pairwise correlation matrices.

Temperature effects on alpha-crystallin structure probed by 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive two-wavelength fluorescent dye covalently attached to the single Cys residue.

The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment.

A triterpene oleanolic acid conjugate with 3-hydroxyflavone derivative as a new membrane probe with two-color ratiometric response.

We report on the synthesis by coupling of a triterpenoid oleanolic acid with 4'-diethylamino-3-hydroxyflavone (FE) to produce an environment-sensitive biomembrane probe with two-band ratiometric response in fluorescence emission. The synthesized compound (probe FOT) was tested in a series of model solvents and demonstrated the response to solvent polarity and intermolecular hydrogen bonding very similar to that of parent probe FE. Meantime when incorporated into lipid bilayer membranes, it showed new features differing in response between lipids of different surface charges as well as between glycerophospholipids and sphingomyelin.

Revisiting the armed-disarmed concept rationale: s-benzoxazolyl glycosides in chemoselective oligosaccharide synthesis.

[reaction: see text]. It has been discovered that 2-O-benzyl-3,4,6-tri-O-acyl SBox glycosides are significantly less reactive than even "disarmed" peracylated derivatives. This finding has been applied to the synthesis of various oligosaccharides, the monomeric units of which are connected via cis-cis, trans-cis, and cis-trans sequential glycosidic linkages.

The future of fluorescence sensor arrays.

The rapid progress in sensor and biosensor array technologies needs a general strategy in the design of fluorescence reporters. Such reporters should provide a high density of sensor elements, allow analysis of targets of different affinities, and be internally calibrated, reproducible and have a rapid readout. Several criteria are introduced here for the comparative evaluation of fluorescence-sensing techniques.

Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes.

The dipole potential (Psi(d)) constitutes a large and functionally important part of the electrostatic potential of cell plasma membranes. However, its direct measurement is not possible. Herein, new 3-hydroxyflavone fluorescent probes were developed that respond strongly to Psi(d) changes by a variation of the intensity ratio of their two well-separated fluorescence bands.

The binding of novel two-color fluorescence probe FA to serum albumins of different species.

The novel two-color ratiometric fluorescence probe FA belonging to a class of 3-hydroxychromone dyes was applied for characterization of binding sites in serum albumins obtained from seven species (bovine, dog, horse, human, pig, rabbit and sheep). On strong and highly specific FA binding to the same location in protein structure, the species-dependent differences were observed in positions of absorption maxima, positions of two fluorescence emission bands and the intensity ratios between them. They were analyzed by multiparametric algorithm that allowed a detailed characterization of probe-binding sites and were characterized by very low polarity, high electronic polarizability and different extent of intermolecular hydrogen bonding.

Heat perturbation of bovine eye lens alpha-crystallin probed by covalently attached ratiometric fluorescent dye 4'-diethylamino-3-hydroxyflavone.

Bovine eye lens alpha-crystallin was covalently labeled with 6-bromomethyl-4'-diethylamino-3-hydroxyflavone and studied under native-like conditions and at the elevated temperature (60 degrees C) that is known to facilitate alpha-crystallin chaperone-like activity. This novel SH-reactive two-band ratiometric fluorescent probe is characterized by two highly emissive N*- and T*-bands; the latter appears due to excited state intramolecular proton transfer reaction. The positions of these bands and the ratio of their intensities for the alpha-crystallin-dye conjugate are the sensitive indicators of polarity of the dye environment and its participation in intermolecular hydrogen bonding.

Antibody immobilisation on the metal and silicon surfaces. The use of self-assembled layers and specific receptors.

The use of Staphylococcal protein A and lectins as intermediate immobilising agents allows operators to orient antibodies (Ab) towards the solution due to the presence of a specific binding sites of immunoglobulin (Ig) molecules. Antibodies of different species of animals have unequal affinities to individual lectins. The effective thickness of immobilised Ab's depends on the type of substrates used and increases in the following sequence: bare gold or silicon surface, the surface treated with self-assembled polyelectrolytes (PESA) or with protein A or some lectins deposited on the preliminary formed polyelectrolyte layer.

[Immunoenzyme analysis of non-ionic surfactants in water].

The approaches for high sensitive and specific determination of nonylphenol (NP) with the help of immune enzymatic (ELISA) method have been developed. The process of preparation of conjugates of NP with proteins, antiserum obtaining, purification of immunoglobulin (Ig) fractions and study of specificity of the obtained antibodies were described in detail. It was shown that the antiserum and total Ig fraction do not differ in respect of specificity and binding abilities.

A novel strategy for oligosaccharide synthesis via temporarily deactivated S-thiazolyl glycosides as glycosyl acceptors.

A new glycosylation strategy that allows chemoselective activation of the S-thiazolyl (STaz) moiety of a glycosyl donor over the temporarily deactivated glycosyl acceptor, bearing the same anomeric group, has been developed. This deactivation is achieved by engaging of the STaz moiety of the glycosyl acceptor into a stable palladium(II) complex. Therefore, obtained disaccharides are then released from the complex by simple ligand exchange.

Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes.

The penetration of water into the hydrophobic interior leads to polarity and hydration profiles across lipid membranes which are fundamental in the maintenance of membrane architecture as well as in transport and insertion processes into the membrane. The present paper is an original attempt to evaluate simultaneously polarity and hydration properties of lipid bilayers by a fluorescence approach. We applied two 3-hydroxyflavone probes anchored in lipid bilayers at a relatively precise depth through their attached ammonium groups.

Resolution of Cys and Lys labeling of alpha-crystallin with site-sensitive fluorescent 3-hydroxyflavone dye.

Ratiometric fluorescent probes based on 3-hydroxyflavone (3HF) are highly sensitive tools for studying polarity, hydration, electronic polarizability, and electrostatics in different microheterogeneous systems, including protein molecules. In the present work, a reactive derivative of 3HF, 6-bromomethyl-4'-diethylamino-3-hydroxyflavone, recently synthesized in our group, was applied to label covalently bovine lens alpha-crystallin. The labeling of SH and NH(2) groups are clearly distinguished by spectroscopic criteria.

Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers.

A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer.

Towards realistic description of collective motions in the lattice protein folding models.

Collective motions and the formation of clusters of residues play an important role in the folding of real proteins. However, existing Monte Carlo (MC) techniques of the protein folding simulations based on highly popular lattice models provide only a schematic representation of collective motions, which is rather far from physical reality. The Clustering Monte Carlo (CMC) algorithm was developed with particular aim to provide a realistic description of collective motions on the lattice.

Quaternary structure of alpha-crystallin is necessary for the binding of unfolded proteins: a surface plasmon resonance study.

The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein.

Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes.

The principle of electrochromic modulation of excited-state intramolecular proton-transfer reaction was applied for the design of fluorescence probes with high two-color sensitivity to dipole potential, Psid, in phospholipid bilayers. We report on the effect of Psid variation on excitation and fluorescence spectra of two new 3-hydroxyflavone probes, which possess opposite orientations of the fluorescent moiety in the lipid bilayer. The dipole potential in the bilayer was modulated by the addition of 6-ketocholestanol or phloretin and by substitution of dimyristoyl phosphatidylcholine lipid with its ether analog 1,2-di-o-tetradecyl-sn-glycero-3-phosphocholine, and its value was estimated by the reference styryl dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine.

Synthesis and biological evaluation of Rhizobium sin-1 lipid A derivatives.

A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed. The approach employed the advanced intermediate 3-O-acetyl-6-O-(3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-d-glucopyrano-syl)-2-azido-4-O-benzyl-2-deoxy-1-thio-alpha-d-glucopyranoside (5), which is protected in such a way that the anomeric center, the C-2 and C-2' amino groups, and the C-3 and C-3' hydroxyls can be selectively functionalized. The synthetic strategy was used for the preparation of 2-deoxy-6-O-[2-deoxy-3-O-[(R)-3-hydroxy-hexadecanoyl]-2-[(R)-3-octacosanoyloxy-hexadecan]amido-beta-d-glucopyranosyl]-2-[(R)-3-hydroxy-hexadecan]amido-3-O-[(R)-3-hydroxy-hexadecanoyl]-alpha-d-glucopyranose (11) and 2-deoxy-6-O-[2-deoxy-3-O-[(R)-3-hydroxy-hexadecanoyl]-2-[(R)-3-octacosanoyloxy-hexadecan]amido-beta-d-glucopyranosyl]-2-[(R)-3-hydroxy-hexadecan]amido-3-O-[(R)-3-hydroxy-hexadecanoyl]-d-glucono-1,5-lactone (13), which contain an unusual octacosanoic acid moiety and differ in the oxidation state of the anomeric center.

Solvent influence on excited-state intramolecular proton transfer in 3-hydroxychromone derivatives studied by cryogenic high-resolution fluorescence spectroscopy.

High-resolution Shpol'skii spectra (recorded at 10 K in n-octane) of 3-hydroxychromone (3HC) substituted at the 2-position with a furan (3HC-F), a benzofuran (3HC-BF) or a naphthofuran group (3HC-NF) are presented. Being close analogues of 3-hydroxyflavone (3HF), these compounds can undergo excited-state intramolecular proton transfer (ESIPT). Luminescence can occur from the normal N* state (blue) or from the tautomeric T* state (green).

Novel two-color fluorescence probe with extreme specificity to bovine serum albumin.

We report on strong, highly specific and stochiometric binding to bovine serum albumin of novel fluorescence probe FA, 2-(6-diethylaminobenzo[b]furan-2-yl)-3-hydroxychromone, that exhibits a very characteristic two-band fluorescence spectrum. Both absorption band and two fluorescence bands of FA are very sensitive to non-covalent interactions in the immediate environment of the probe. Multiparametric analysis of this spectroscopic information allows us to conclude that the binding site is characterized by very low polarity, high extent of screening from aqueous environment and unusually high electronic polarizability.

Perturbation of planarity as the possible mechanism of solvent-dependent variations of fluorescence quantum yield in 2-aryl-3-hydroxychromones.

In order to understand the unexpectedly low quantum yields of 3-hydroxyflavones (3-HFs) in certain solvents, such as acetonitrile or ethyl acetate, the comparative study of solvent-dependent properties of parent 3-HF, 2-furyl-3-hydroxychromone and 2-benzofuryl-3-hydroxychromone derivatives have been performed. The results suggest that the formation of intermolecular hydrogen bond of 3-hydroxy group with the solvent favors non-planar conformations of phenyl group with respect to chromone system. This steric hindrance is not observed in the case of furan- and benzofuran-substituted 3-hydroxychromones (3-HCs).

S-Benzoxazolyl (SBox) glycosides as novel, versatile glycosyl donors for stereoselective 1,2-cis glycosylation.

[reaction: see text] Novel glycosyl donors, S-benzoxazolyl (SBox) glycosides, have been synthesized, tested toward various protecting group manipulations, and applied to the highly stereoselective 1,2-cis glycosylation. These compounds fulfill the requirements for a modern glycosyl donor such as accessibility, high stability toward protecting group manipulations, and mild activation conditions. It was also demonstrated that SBox glycosides withstand other glycosyl donor activation conditions and therefore allow selective glycosylations of O-pentenyl and thioglycosides.