Peter Nikolov

Regulation of the Inner Membrane Mitochondrial Permeability Transition by the Outer Membrane Translocator Protein (Peripheral Benzodiazepine Receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca2+ uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.

Inclusion of 5-[4-(1-Dodecanoylpyridinium)]-10,15,20-triphenylporphine in Supramolecular Aggregates of Cationic Amphiphilic Cyclodextrins: Physicochemical Characterization of the Complexes and Strengthening of the Antimicrobial Photosensitizing Activity

Recent findings suggest that visible light-promoted photooxidative processes mediated by sensitizers of appropriate chemical structure could represent a useful tool for properly addressing the problem of the increasing occurrence of infectious diseases caused by multiantibiotic-resistant microbial pathogens. The monocationic meso-substituted porphyrin 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphine (TDPyP) complexed into supramolecular aggregates of cationic amphiphilic beta-cyclodextrin (SC(6)NH(2)) (mean diameter = 20 nm) appeared to be endowed with favorable properties to act as a photosensitizing agent, including a very high quantum yield (Phi(Delta) = 0.90) for the generation of the highly reactive oxygen species, singlet oxygen ((1)O(2)). Although the yield of (1)O(2) generation was comparable to that obtained after TDPyP incorporation into cationic unilamellar liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) SC(6)NH(2)-bound TDPyP was more active than DOTAP-bound TDPyP in photosensitizing the inactivation of the Gram-positive methicillin-resistant bacterium Staphylococcus aureus (MRSA). At variance with DOTAP-bound TDPyP, photoactivated SC(6)NH(2)-bound TDPyP was efficient also in photokilling Gram-negative bacterial pathogens, such as Escherichia coli . These observations are in agreement with the well-known photobactericidal effect of positively charged porphyrin derivatives, which can be markedly enhanced after incorporation into carriers with multiple positive charges. In addition, transmission electron microscopy studies revealed that potentiation of the TDPyP-mediated photobactericidal effect by incorporation into SC(6)NH(2) is a consequence of the carriers ability to promote an efficient crossing of the very tightly organized three-dimensional architecture of the bacterial outer wall by the embedded porphyrin so that a prompt interaction between the short-lived photogenerated (1)O(2) and the nearby targets, whose integrity is critical for cell survival, can take place.

Tuning the Excited-State Dynamics of GFP-Inspired Imidazolone Derivatives

The excited-state dynamics of five derivatives of the GFP-chromophore, which differ by the position and nature of their substituents, has been investigated in solvents of various viscosity and polarity and in rigid media using femtosecond-resolved spectroscopy. In polar solvents of low viscosity, like acetonitrile or methanol, the fluorescence decays of all compounds are multiexponential, with average lifetimes of the order of a few picoseconds, whereas in rigid matrices (polymer films and low temperature glasses), they are single exponential with lifetimes of the order of a few nanoseconds and fluorescence quantum yields close to unity. Global analysis of the fluorescence decays recorded at several wavelengths and of the transient absorption spectra reveals the presence of several excited-state populations with slightly different fluorescence and absorption spectra and with distinct lifetimes. These populations are attributed to the existence of multiple ground-state conformers. From the analysis of the dependence of the excited-state dynamics on the solvent and on the nature of the substituents, it follows that the nonradiative deactivation of all these excited chromophores involves an intramolecular coordinate with large amplitude motion. However, depending on the solvent and substituent, additional channels, namely, inter- and intramolecular hydrogen bond assisted nonradiative deactivation, are operative. This allows tuning of the excited-state lifetime of the chromophore. Finally, an ultrafast photoinduced intramolecular charge transfer is observed in polar solvents with one derivative bearing a dimethylaminophenyl substituent.

Dimer fluorescence of some β-dicarbonyl compounds

Abstract Luminescence and absorption spectra of β-dicarbonyl compounds have been studied over the concentration range 10−7–10−2 M. The temperature dependence of the emission maxima was followed from 10 to 70°C. The emission and long-wave absorption at high concentrations is related to dimers of the enol tautomer. The association constant and change in free energy are evaluated.

Interaction of phthalocyanines with lipid membranes: a spectroscopic and functional study on isolated rat liver mitochondria

Absorption and emission spectroscopic studies on Zn(II)-phthalocyanine (ZnPc) incorporated into unilamellar liposomes of dipalmitoylphosphatidylcholine, sometimes added with cholesterol or cardiolipin, and released to rat liver mitochondria via the three types of liposomal vesicles indicated that: (a) ZnPc predominantly dissolves in all lipid domains of biological membranes with the exception of cardiolipin-containing regions; a partial localization of ZnPc in protein binding sites is also postulated; (b) the spectroscopic properties of ZnPc, although mainly determined by the aggregation state of the dye, are somewhat influenced by the physico-chemical characteristics of the lipid environment; (c) ZnPc-binding lipid domains in mitochondria are mainly localized in the outer membrane; this conclusion is clearly deduced from the trends of Arrhenius plots of the ZnPc fluorescence quantum yield in whole mitochondria and isolated inner or outer membrane in the temperature range -10 degrees C-(+)45 degrees C; (d) the nature of the ZnPc-binding site in mitochondria is not dependent on the chemical composition of the liposome carrier, contrary to what observed for other hydrophobic dyes, such as porphyrins. This has been also confirmed by photosensitization experiments. Actually, illumination of ZnPc-loaded mitochondria by 600-700 nm light causes a decline of the respiratory control ratio, which is essentially dependent on the amount of incorporated photosensitizer, irrespective of the composition of the carrier.

DEACTIVATION PROCESSES AND HYDROGEN BONDING OF EXCITED N-SUBSTITUTED ACRIDONES

Abstract The photophysical properties of 10-decyl- and 10-propenyl-9-acridones, important emitters in electro- and chemiluminescence, are investigated in dependence of the solvent polarity and its hydrogen bonding ability at room temperature as well as in frozen matrix at 77 K. Fluorescence is inefficient in non-polar solvents at room temperature due to fast intersystem crossing but increases with solvent polarity due to the inversion of the S 1 (ππ ∗ ) and the T (nπ ∗ ) states. In the case of 9-[10-(1-propenyl)]-acridone the dependencies of the fluorescence spectrum and quantum yield on the ET(30) solvent parameters indicate that intermolecular hydrogen bonded solute-solvent complexes are formed in the excited state, inducing efficient non-radiative deactivation.

Photophysical properties and quantum chemical calculations of differently substituted 2(2-phenylethenyl)-benzoxazoles and benzothiazoles

The low fluorescence quantum yield of 2(2-phenylethenyl)-benzoxazoles and benzothiazoles in solution at room temperature is attributed to intramolecular librations with great amplitude of the two heavy fragments at the both ends of the allyl chain. Freezing the solutions at 77 K leads to remarkable increase of fluorescence quantum yield up to three orders of magnitude. No phosphorescence at 77 K is observed. The analysis of the results from quantum-chemical calculations (AM1 approximation) supports the hypothesis for the quenching processes in excited sate.

Excimer fluorescence from acridine and diaza-heterocyclic hydrocarbons in non-polar media at low temperatures

Abstract The fluorescence of excited acridine, benzo[b][1,6]naphthyridine (N), 10-phenyl-benzo[b][1,6]naphthyridine (PN) and a series of 6- and 8-substituted derivatives was studied by stationary and transient techniques in two non-polar solvents: methylcyclohexane and a mixture of 2,2′-dimethylbutane and n-pentane. The emission spectrum observed at 77 K for all the investigated compounds using concentrations of 0.03–0.5 mM, in comparison with the fluorescence spectrum at room temperature, is dominated by a red-shifted structureless band with a maximum above 500 nm, i.e. 500 nm for acridine, 515 nm for N and 540 nm for parent PN. Owing to the lifetimes of 40–80 ns and the concentration dependence of the two bands, this new emission is attributed to excimer fluorescence. The quantum yields of excimer fluorescence are in the range ΦfE = 0.01–0.3, depending on the structure and substituent. Although ΦfE is largest at 77 K, the excimer fluorescence can be examined in most cases up to approximately 200 K. This shows that a glass is not necessary for excimer formation. The photophysical processes, the effect of temperature and some mechanistic consequences are discussed.

Fluorescence Studies on Denaturation and Stability of Recombinant Human Interferon-Gamma

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNγ) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 m and ΔG0 = 13.4 kJ/mol) than urea (C* = 2.8 m and ΔG0 = 11.7 kJ/mol). The close ΔG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNγ is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNγ remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNγ, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNγ.