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Biomedical subjects

Alexander P Demchenko

Publications and source records attributed to Alexander P Demchenko.

At least 19 recordsLinked to original sources
PubMed2006

Dramatic pressure-dependent quenching effects in supercritical CO2 assessed by the fluorescence of 4'-dimethylamino-3-hydroxyflavone. Thermodynamic versus kinetics control of excited-state intramolecular proton transfer.

Steady-state fluorescence of 4'-dimethylamino-3-hydroxyflavone (DMA3HF) was observed in supercritical carbon dioxide (scCO(2)). Excited-state intramolecular proton transfer (ESIPT) occurs resulting in two well-separated emission bands corresponding to the normal and tautomer forms. As the scCO(2) density exceeds 0.7 g/mL, the relative intensity of the two bands tends to a constant value, comparable to that observed for organic solvents with ET(30) = 33.0 +/- 0.5 kcal/mol, such as toluene and di-n-butyl ether. At lower densities, the substantial decrease of the total fluorescence intensity (a 600-fold decrease as the pressure decreases from 100 to 80 bar) is accompanied by an even more accentuated decrease of the tautomer fluorescence. This can be explained by a shift in the equilibrium between normal and tautomer forms, concomitant with a more efficient quenching of the less solvated fluorophore, that may change the thermodynamic control of the relative population of the two emissive species to a kinetic control.

Carbon Dioxide↗
PubMed2006

The change of protein intradomain mobility on ligand binding: is it a commonly observed phenomenon?

Analysis of changes in the dynamics of protein domains on ligand binding is important in several aspects: for the understanding of the hierarchical nature of protein folding and dynamics at equilibrium; for analysis of signal transduction mechanisms triggered by ligand binding, including allostery; for drug design; and for construction of biosensors reporting on the presence of target ligand in studied media. In this work we use the recently developed HCCP computational technique for the analysis of stabilities of dynamic domains in proteins, their intrinsic motions and of their changes on ligand binding. The work is based on comparative studies of 157 ligand binding proteins, for which several crystal structures (in ligand-free and ligand-bound forms) are available. We demonstrate that the domains of the proteins presented in the Protein DataBank are far more robust than it was thought before: in the majority of the studied proteins (152 out of 157), the ligand binding does not lead to significant change of domain stability. The exceptions from this rule are only four bacterial periplasmic transport proteins and calmodulin. Thus, as a rule, the pattern of correlated motions in dynamic domains, which determines their stability, is insensitive to ligand binding. This rule may be the general feature for a vast majority of proteins.

Biosensing Techniques↗
PubMed2006

Dynamic protein domains: identification, interdependence, and stability.

Existing methods of domain identification in proteins usually provide no information about the degree of domain independence and stability. However, this information is vital for many areas of protein research. The recently developed hierarchical clustering of correlation patterns (HCCP) technique provides machine-based domain identification in a computationally simple and physically consistent way. Here we present the modification of this technique, which not only allows determination of the most plausible number of dynamic domains but also makes it possible to estimate the degree of their independence (the extent of correlated motion) and stability (the range of environmental conditions, where domains remain intact). With this technique we provided domain assignments and calculated intra- and interdomain correlations and interdomain energies for >2500 test proteins. It is shown that mean intradomain correlation of motions can serve as a quantitative criterion of domain independence, and the HCCP stability gap is a measure of their stability. Our data show that the motions of domains with high stability are usually independent. In contrast, the domains with moderate stability usually exhibit a substantial degree of correlated motions. It is shown that in multidomain proteins the domains are most stable if they are of similar size, and this correlates with the observed abundance of such proteins.

Computer Simulation↗
PubMed2006

Visualization and sensing of intermolecular interactions with two-color fluorescent probes.

We developed a new generic fluorescence sensing technology based on the change of relative intensities between two well-separated emission bands of the novel functional 3-hydroxychromone (3HC) dyes. A greatly enhanced self-calibrating wavelength-ratiometric response is obtained to all major types of non-covalent interactions that can be used in sensing--to polarity, hydrogen bonding ability and to local electrostatic fields. This technology may find a broad range of applications--from homogeneous assays in solutions to microarrays, microfluidic devices, nanosensors and whole cell imaging systems. It allows transforming micelles or phospholipid vesicles into nanosensor devices. In cellular research a high sensitivity to membrane potentials can be obtained and the membrane changes during apoptosis detected.

Biosensing Techniques↗
PubMed2006

The effects of thermal quenching on the excited-state intramolecular proton transfer reaction in 3-hydroxyflavones.

The 3-hydroxyflavone (3HF) and its derivatives are the classical objects in the studies of the mechanisms of excited-state intramolecular proton transfer (ESIPT) reaction due to very frequent observation of two separate bands in fluorescence emission belonging to reactant and reaction product. Those of them possessing electron-donor groups in 4' position find many applications as fluorescence sensors and probes because of their much higher sensitivity of their two-band ratiometric response to interactions with the environment. We report on the strong differences between 3HF and such derivatives in the behavior of their fluorescence spectra as a function of temperature. The thermal quenching changes the intensity ratio of two bands strongly for 3HF but does not change it for its studied derivatives. These results are interpreted in terms of different kinetic mechanisms of ESIPT reaction. In 3HF the equilibrium between the two excited-state species is not established prior to emission, so that the ESIPT reaction is under kinetic control, but in these derivatives the equilibrium is established faster than the emission and the reaction is under thermodynamic control. We suggest that the thermal perturbation of fluorescence spectra can be an extremely simple and convenient alternative to time-resolved spectroscopy for determining if slow irreversible or fast reversible ESIPT reaction gives rise to two bands of fluorescence spectra of similar magnitude. This is essential for the development of new wavelength-ratiometric fluorescence sensors and probes.

Flavonoids↗
PubMed2005

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. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate alpha-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3 h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of alpha-crystallin quaternary structure. Since there is only one Cys residue in alphaA subunit of alpha-crystallin (whereas alphaB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye's microenvironment to the particular site within alpha-crystallin molecule.

Animals↗
PubMed2005

The problem of self-calibration of fluorescence signal in microscale sensor systems.

Most recent developments in fluorescent molecular sensor devices are based on "ON-OFF" switching, which is an operation with a single measurable parameter, commonly the quenching of total intensity. In the meantime, with this approach self-calibration on the molecular level is not achievable. This calibration is strongly needed in all kinds of microscale applications, including microarrays, microfluidic systems and living cell imaging. Different possibilities are discussed for overcoming this difficulty and an "OR-OR" switching concept is suggested that involves the two-channel detection as a promising solution. For achieving the desired efficiency specific conditions are needed: it should be a single reporter dye exhibiting rapid reversible excited-state reaction and providing two-band wavelength ratiometric response.

Biosensing Techniques↗
PubMed2005

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. We observed that in the conditions of coexistence of rafts and non-raft structures the probe is excluded from the rafts.

Flavonoids↗
PubMed2005

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. Using fluorescence spectroscopy with cell suspensions and confocal microscopy with adherent cells, we showed, for the first time, two-color fluorescence ratiometric measurement and visualization of Psi(d) in cell plasma membranes. Using this new tool, a heterogeneous distribution of this potential within the membrane was evidenced.

Animals↗
PubMed2005

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. The species-dependent differences were also observed in time-resolved fluorescence emission decays. Fluorescence competition experiments with the drug warfarin, suggested the location of FA binding site within or in proximity to Domain IIA.

Algorithms↗
PubMed2005

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. It is shown that only the two-band wavelength ratiometric sensing with a single reporter dye exhibiting rapid reversible excited-state reaction can satisfy all these criteria and is a prospective candidate for further development.

Biosensing Techniques↗
PubMed2004

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. They are present in two forms: either in H-bond-free form displaying a two-band emission due to an excited state intramolecular proton transfer reaction (ESIPT), or in H-bonded form displaying a single-band emission with no ESIPT. The individual emission profiles of these forms were obtained by deconvolution of the probes' fluorescence spectra. The polarity of the probe surrounding the bilayer was estimated from the two-band spectra of the H-bond-free form, while the local hydration was estimated from the relative contribution of the two forms. Our results confirm that by increasing the lipid order (phase transition from fluid to gel phase, addition of cholesterol or decrease in the lipid unsaturation), the polarity and to a lesser extent, the hydration of the bilayers decrease simultaneously. In contrast, when fluidity (i.e. lipid order) is kept invariant, increase of temperature and of bilayer curvature leads to a higher bilayer hydration with no effect on the polarity. Furthermore, no correlation was found between dipole potential and the hydration of the bilayers.

Flavonoids↗
PubMed2004

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. We observe that the NH(2) labeling creates the positive charge in the proximity to fluorophore, which results in strong internal Stark effect producing the shift in excitation spectrum by ca. 15 nm. Analysis of excitation-dependent fluorescence spectra allows separation of the emission profiles of these SH- and NH(2)-labeled species. Applying recently developed multiparametric analysis of the obtained emission spectra, we described the physicochemical properties of the sites of SH and NH(2) labeling in alpha-crystallin. The site of SH labeling has medium-low polarity (dielectric constant, epsilon = 4.9 +/- 0.9) is protic, and does not contain proximal aromatic residues (according to the obtained refractive index, n = 1.41 +/- 0.14). The site of NH(2) labeling is also of medium-low polarity. The novel label due to its two-wavelength ratiometric response and high sensitivity to the type of labeling may offer new possibilities in the studies of structure, dynamics, and interactions of proteins by probing their SH- and NH(2)-labeling sites.

Cysteine↗
PubMed2004

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. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn(1)-carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.

Algorithms↗
PubMed2004

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. Our data indicate that quaternary structure of alpha-crystallin is necessary for its chaperone-like activity.

Animals↗
PubMed2003

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. We demonstrate that after Psid changes, the probe orienting in the bilayer similarly to the reference dye shows similar shifts in the excitation spectra, whereas the probe with the opposite orientation shows the opposite shifts. The new observation is that the response of 3-hydroxyflavone probes to Psid in excitation spectra is accompanied by and quantitatively correlated with dramatic changes of relative intensities of the two well separated emission bands that belong to the initial normal and the product tautomer forms of the excited-state intramolecular proton-transfer reaction. This provides a strong response to Psid by change in emission color.

Fluorescent Dyes↗
PubMed2003

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. The latter suggests the proximal location of probe FA to the aromatic amino acid residues in the binding site. The new probe can be proposed for the study of interaction of ligands and drugs of different nature with serum albumins.

Chromones↗