Alexander P. Demchenko

Recognition between flexible protein molecules: induced and assisted folding†

This review focuses on a very important but little understood type of molecular recognition — the recognition between highly flexible molecular structures. The formation of a specific complex in this case is a dynamic process that can occur through sequential steps of mutual conformational adaptation. This allows modulation of specificity and affinity of interaction in extremely broad ranges. The interacting partners can interact together to form a complex with entirely new properties and produce conformational signal transduction at substantial distance. We show that this type of recognition is frequent in formation of different protein–protein and protein–nucleic acid complexes. It is also characteristic for self‐assembly of protein molecules from their unfolded fragments as well as for interaction of molecular chaperones with their substrates and it can be the origin of ‘protein misfolding’ diseases. Thermodynamic and kinetic features of this type of dynamic recognition and the principles underlying their modeling and analysis are discussed. Copyright

A 3-hydroxychromone with dramatically improved fluorescence properties

A new 3-hydroxychromone derivative, 2-(6-diethylaminobenzo(b)furan-2-yl)-3-hydroxychromone, has been synthesized by a concise route. Possessing dual emission common for 3-hydroxyflavones, it exhibits strong red shifts of both absorption and fluorescence spectra, which makes it the longest wavelength fluorescent dye among all known chromones. It also demonstrates a significant increase in fluorescence quantum yield in aprotic solvents and shift in solvent-polarity-dependent switch between normal and tautomer emissive forms. This derivative offers new possibilities in designing novel molecular sensors.

Nanosecond dynamics of charged fluorescent probes at the polar interface of a membrane phospholipid bilayer

Molecular relaxation fluorescence methods were applied to analyze the nature and characteristic times of motions of amphiphilic molecules absorbed in the polar region of a phospholipid bilayer. The fluorescence probes 2-toluidinonaphthalene-6-sulfonate and 1-anilinonaphthalene-8-sulfonate in egg phosphatidylcholine vesicles were studied. The methods of edge excitation fluorescence red shifts, nanosecond time-resolved spectroscopy, fluorescence quenching by hydrophilic and hydrophobic quenchers and emission wavelength dependence of polarization were used. The structural (dipolar) relaxation is shown to be a very rapid (subnanosecond) process. The observed nanosecond phenomena are related to translational movement of the chromophore itself towards a more polar environment and its rotation. The polar surface area of the phospholipid membrane appears to be a highly mobile liquid-like system.

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). These results suggesting a new strategy for dramatic improvement of fluorescence properties of 3-HCs as two-wavelength ratiometric fluorescence probes.

Bands separation in fluorescence spectra of ketocyanine dyes: evidence for their complex formation with monohydric alcohols

Abstract In the studies of binary solvent systems containing non-polar (toluene) and polar proton-donating components (monohydric alcohols) using ketocyanine dyes of 2,5-di-benzylidene-cyclopentanone-1 type as solvent polarity probes, we found that in addition to common solvent polarity-dependent shifts of fluorescence spectra, at low alcohol concentrations there appear two new well-resolved spectral bands. They are attributed to the emission of hydrogen bonded complexes of 1:1 and 1:2 type. Effective constants for hydrogen bond complex formation were estimated for them from the fluorescence titration data.

Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease.

The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.

Selective laser spectroscopy of 1-phenylnaphthylamine in phospholipid membranes.

Time-resolved and steady-state spectra and kinetics of anisotropy of 1-phenylnaphthylamine (1-AN) fluorescent probe in phosphatidylcholine bilayer membranes have been examined using a nanosecond laser spectrofluorimeter. In this system we consider two kinds of inhomogeneous broadening of spectra, the first of which is due to different probe locations in membrane, while the second one is due to the statistical distribution of interaction energy within a given location. This broadening causes the red shift of the fluorescence spectrum at red-edge excitation, the specific dependences of instantaneous fluorescence and fluorescence anisotropy spectra on the wavelength of excitation. A field diagram is presented which, by describing the free energy levels of the polar fluorescent probe in membranes, makes it possible to unambiguously interpret the whole set of experimental data. It is suggested that the release of potential energy of intermolecular interactions which occurs in the process of relaxation, results in accelerated (light-induced) rotation of the probe inside the membrane.

Ratiometric Probes: Design and Applications

With the aim of substantial improvement of solvatochromic and electrochromic fluorescence probes by coupling their response with an excitedstate reaction, a series of 3-hydroxychromone derivatives have been synthesized. They demonstrate two well-separated emission bands that originate from normal and phototautomer forms due to excited-state intramolecular proton transfer (ESIPT) reaction. In the studies of solvent polarity effect, electrochromism (internal Stark effect) and red edge effect a strong amplification of probe response is achieved by recording of relative intensity ratios of these fluorescence bands instead of their spectral shifts. This result is an illustration of a new principle in design of fluorescence probes, which may allow the achievement of almost perfect on-off switching behavior.

Photophysics of dimethylamino-substituted polymethine dye in polymeric media

Abstract The photophysical mechanism responsible for the large Stokes shift in dimethylamino-substituted polymethine dye (DMA-PD) has been studied by steady-state and time-resolved fluorescence spectroscopy. Experiments in low-temperature ethanol glass and in polymer polyurethane acrylate (PUA) in glassy and elastic states allow us to conclude that the emission occurs mainly from two excited states with lifetimes of 2–3 and 6–7 ns, respectively. The excited-state dynamics includes fast subnanosecond rotation of the DMA group around the carbon–dimethylamino bond of the dye molecule and stabilization of this form due to environmental rearrangement. The large Stokes shift remains for this dye in solid media. This process is dynamic even in polymeric media, where it occurs in the subnanosecond–nanosecond time scale. The major spectral shift can be described as an intramolecular excited-state reaction affecting either or both emissive species. We suggest a model in which the twisting of the small dimethylamino group modulates the effective length of the polymethine chromophore, producing the spectral shift. In addition, time-resolved fluorescence anisotropy decays show a slow rotational motion (about 40 ns) of the dye inside the polymer microcavities. These interesting features of the spectral behavior of the PUA matrix doped by DMA-PD may find various applications.

Complex formation between azacrown derivatives of dibenzylidenecyclopentanone and alkali-earth metal ions

The complex formation of 2,5-bis[4-(1,7,10,13-tetraoxa-4-azacyclopentadec-4-yl)benzylidene]cyclopentanone and several model compounds, prospective metal-sensitive fluorescent probes, with Mg2+, Ca2+, and Ba2+ ions in acetonitrile was studied. The azacrown derivatives of dibenzylidenecyclopentanone have two complex formation centers, azacrown cycle and carbonyl group. The sequence of binding to these sites is different for different ions. The efficient ejection of the Ca2+ and Ba2+ ions from their complexes with azacoronands was observed in the excited state, whereas in the case of the Mg2+ ion, this process occurred only partially.