Optically detected zero field magnetic resonance studies of proflavine-DNA and 3,4-benzpyrene-DNA complexes.

Optical detection of magnetic resonance has been used to observe the photoexcited triplet state zero-field transitions of proflavine in DNA and 3,4-benzpyrene in DNA at 2°K. The results suggest that optically detected magnetic resonance may be utilized for determining the local site distribution of small molecules bound to DNA.     

Extrinsic cotton effects of aminoacridines bound to DNA

The optical rotatory dispersion curves of the proflavine cation were measured in the spectral range 400–500 mμ. No optical activity was observed for the free cation but a large positive Cotton effect appeared in the presence of DNA. The effect of ionic strength, denaturation of the DNA, and the DNA/proflavine ratio were studied. The dependence of the magnitude of the Cotton effect on the DNA/proflavine ratio suggests that a nearest-neighbor interaction between bound proflavine molecules is necessary for optical activity. A simple statistical treatment was made which indicated that only a small number of proflavine molecules are required in close proximity for optical activity to occur. Denaturation of the DNA did not destroy the optical activity, which shows that long runs of DNA double helix are not necessary for optical activity of the ligand molecules. The optical rotatory dispersion curves of acridine orange which was bound to DNA were also measured. Two Cotton effects of opposite sense could be distinguished, the relative magnitudes of which depended on the DNA/acridine orange ratio and the state of denaturation of the DNA. The apparent differences from the proflavine-DNA system can to a large extent be explained in terms of the tendency of acridine orange to form aggregates.     

Extrinsic Cotton effects of proflavine bound to polynucleotides

The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3–4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.     

Triplet state quenching by photoinduced radicals in the sensitized ethanol photolysis

UV irradiation of a rigid solution of 2-amino-pyrimidine in ethanol at 77 degrees K results in a sensitized production of ethyl radicals. The radicals are formed via a biphotonic process. The increase of the radical yield with irradiation time is directly correlated to a decrease of the number of triplet state molecules as detected by ESR. A kinetic model of the biphotonic process is presented and verified by experimental results. It supports the hypothesis of a catalytically enhanced radiationless decay process of the triplet states due to an interaction between the radicals and the triplet state molecules.     

Singlet-triplet excitation fission in light-harvesting complexes of photosynthetic bacteria and in isolated carotenoids

Time-resolved electron paramagnetic resonance was used to study the properties of carotenoid triplet states populated in LH2 light-harvesting complexes of phototrophic bacteria Allochromatium minutissimum, Rhodopseudomonas palustris, and in carotenoid films free of bacteriochlorophyll. The study was performed on purified LH2 preparations not contaminated by reaction centers, and under selective pigment excitation. The obtained results enable a conclusion that the carotenoid triplet states, both in LH2 complexes and films, are populated in the process of homofission of singlet excitation into two triplets, which involves only carotenoid molecules. It is observed that the fission process in magnetic field leads to predominant population of the T₀ spin sublevel of the triplet. One molecular spin sublevel of the triplet is demonstrated to possess an increased probability of intersystem crossing to the ground state, independent of the carotenoid configuration. Pigment composition of the LH2 protein heterodimers is discussed, and a conclusion of the possible presence of two interacting carotenoid molecules is made.     

Spectroscopic properties of the peridinins involved in chlorophyll triplet quenching in high-salt peridinin-chlorophyll a-protein from Amphidinium carterae as revealed by optically detected magnetic resonance, pulse EPR and pulse ENDOR spectroscopies

The photoexcited triplet state of the carotenoid peridinin in the high-salt peridinin-chlorophyll a-protein (HSPCP) of the dinoflagellate Amphidinium carterae was investigated by ODMR (optically detected magnetic resonance), pulse EPR and pulse ENDOR spectroscopies. The properties of peridinins associated to the triplet state formation in HSPCP were compared to those of peridinins involved in triplet state population in the main-form peridinin-chlorophyll protein (MFPCP), previously reported. In HSPCP no signals due to the presence of chlorophyll triplet state have been detected, during either steady-state illumination or laser-pulse excitation, meaning that peridinins play the photo-protective role with 100% efficiency as in MFPCP. The general spectroscopic features of the peridinin triplet state are very similar in the two complexes and allow drawing the conclusion that the triplet formation pathway and the triplet localization in one specific peridinin in each subcluster are the same in HSPCP and MFPCP. However some significant differences also emerged from the analysis of the spectra. Zero field splitting parameters of the peridinin triplet states are slightly smaller in HSPCP and small changes are also observed for the hyperfine splittings measured by pulse ENDOR and assigned to the beta-protons belonging to one of the two methyl groups present in the conjugated chain, (a(iso)=10.3 MHz in HSPCP vs a(iso)=10.6 MHz in MFPCP). The differences are explained in terms of local distortion of the tails of the conjugated chains of the peridinin molecules, in agreement with the conformational data resulting from the X-ray structures of the two complexes.     

Flash photolysis of misonidazole and metronidazole

Laser flash photolysis at 355 nm of misonidazole or metronidazole in aqueous solutions produced the relatively long-lived nitro radical anion as the only observable transient species. When 266 nm excitation was used, a small yield of solvated electron was observed. It is suggested that the nitroimidazole first undergoes photoionization and the photoelectrons are scavenged by ground state nitroimidazole molecules to produce the nitro radical anion. Alternatively, added EDTA or carbonate ion acted as an electron donor to the excited state nitroimidazole molecule, thereby increasing the yield of nitro radical anion. The transient yield from metronidazole was about half that from misonidazole, while the phosphorescence intensity of metronidazole in an ethanol glass was about 20 times that of misonidazole. The misonidazole n, pi* triplet state is more easily reduced than that of metronidazole and, in the presence of an electron donor, the radical anion is postulated to result from electron transfer to the triplet state of the nitroimidazole.     

Study of the interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in aqueous solution by uni- and two-dimensional 1H-spectroscopy]

Complex formation between acridine dye proflavine and self-complementary deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water-salt solution was studied by the method of one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Two-dimensional homonuclear 1H-NMR spectroscopy (2D-COSY and 2D-NOESY) was used for complete assignments of proton signals of molecules in solution and for qualitative analysis of the nature of interactions between proflavine and tetranucleotide. Concentration dependences of proton chemical shifts of the molecules were measured at 293 K. Equilibrium reaction constants and limiting chemical shifts of dye protons in the complexes were determined using suggested schemes of complex formation. Based on the obtained data possible types of complexes were considered. Analysis of relative content of different types of complexes was made and special features of dynamic equilibrium were revealed as a function of correlation of dye and tetranucleotide concentrations. The most favourable structure of 1:2 complex of dye with tetranucleotide was constructed using the calculated values of induced chemical shifts of proflavine protons and 2D-NOESY spectra.     

Dielectric behavior of DNA-proflavine complex

The dielectric relaxation of namtive DNA and DNA–proflavine complexes at different DNA phosphate (P) to dye (D) ratios, were investigated in the frequency range 100 c/sec to 100 Kc/sec. The proflavine molecules were found to have a profound effect on the static dielectric constant and the relaxation time of the polymers. The static dielectric constant was oberserved to decrese with increasing level of added proflavine. At P/D = 1, the variation of dielectric constant with frequency was small. Relaxation time (τ) was greater for the DNA–proflavine complexes compared to that for free DNA, Maximum value of the relaxation time was obtained at P/D = 10. The increase in the relaxation time and decrease in the static dielectric constant were attributed to the increase in length and meutralization of surface charges of the DNA molecules, respectively, as aresult of proflavine binding.     

Dependence of oxygen quenching of intercalated methylene blue triplet lifetime on DNA base-pair composition

We utilize the dynamic quenching of the triplet state of methylene blue by molecular oxygen to observe changes in the rate at which oxygen can penetrate the helix as a function of base-pair composition. The results indicate that the interior of the oligonucleotide dA-dT is more accessible than dG-dC to small molecules such as dioxygen.     

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluoiescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D = 10. Next, fluorescence decay curves were measured by the monophoton counting technique and analyzed with the aid of the method of moments and the Laplace transform method. The results showed that the fluorescence decay of bound acridine orange was exponential above P/D = 10. On the other hand, the decay of bound proflavine was exponential above P/D = 100, but markedly deviated from exponentiality with decreasing P/D ratio. The results of fluorescence polarization suggested that this phenomenon is the result of Förster energy transfer between proflavine molecules bound to the fluorescent site (AT pair) and bound to the quenching site (GC pair). Critical transfer distances were 26-4 and 37.0 Å, respectively, for bound proflavine and acridine orange.     

Acridine orange interaction with DNA: Effect of ionic strength

BackgroundThe study of acridine orange (AO) spectral characteristics and the quenching of its singlet and triplet excited states by TEMPO radical at its binding to DNA in the function of the DNA concentration and in the absence and presence of NaCl is reported.MethodsThe study was performed using steady-state and time resolved optical absorption and florescence, fluorescence correlation spectroscopy and resonant light scattering techniques.ResultsThe presence of different species in equilibrium: AO monomers and aggregates bound to DNA, has been demonstrated, their relative content depending on the DNA and the AO concentrations. At high DNA concentration the AO monomers are protected against the contact with other molecules, thus reducing the AO excited state quenching. The addition of NaCl reduces the AO binding constant to DNA, thus reducing the AO and DNA aggregation.ConclusionsThe interaction of AO with DNA is a complex process, including aggregation and disaggregation of both components. This modifies the AO excited state characteristics and AO accessibility to other molecules. The salt reduces the DNA effects on the AO excited state characteristics thus attenuating its effects on the AO efficacy in applications.General significanceThis study demonstrates that the interaction of photosensitizers with DNA, depending on their relative concentrations, can both decrease and increase the photosensitizer efficacy in applications. The salt is able to attenuate these effects.     

Isolation of bright aggregate fluctuations in a multipopulation image correlation spectroscopy system using intensity subtraction

Image correlation spectroscopy allows sensitive measurement of the spatial distribution and aggregation state of fluorescent membrane macro molecules. When studying a single population system (i.e., aggregates of similar brightness), an accurate measure can be made of the aggregate number per observation area, but this measurement becomes much more complex in a distributed population system (i.e., bright and faint aggregates). This article describes an alternate solution that involves extraction of the bright aggregate population information. This novel development for image correlation spectroscopy, termed intensity subtraction analysis, uses sequential uniform intensity subtraction from raw confocal images. Sequential intensity subtraction results in loss of faint aggregate fluctuations that are smaller in magnitude than fluctuations due to the brightest aggregates. The resulting image has correlatable fluctuations originating from only the brightest population, permitting quantification of this population's distribution and further cross-correlation measurements. The feasibility of this technique is demonstrated using fluorescent microsphere images and biological samples. The technique is further used to examine the spatial distribution of a plasma-membrane-labeled fluorescent synthetic ganglioside, and to cross-correlate this probe with various membrane markers. The evidence provided demonstrates that bright aggregates of the fluorescent ganglioside are associated with clathrin-coated pits, membrane microvilli, and detergent-resistant membranes.     

Protein rotation, enzyme activity and photooxidation of SH groups in sarcoplasmic reticulum Ca2+-ATPase

The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca2+-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet yield of these probe molecules. Consistent with this, the greatest effect is seen with erythrosin isothiocyanate and the least effect with fluorescein isothiocyanate. These reductions of ATPase activity associated with illumination are also associated with an aggregation of the protein molecules. This is indicated by laser flash photolysis measurements and also by polyacrylamide gel electrophoresis. A reduction in the number of thiol groups present on the ATPase molecule parallels the reduction of enzyme activity and changes in the protein mobility. The results are discussed in relation to the use of these probe molecules to study biological systems and also in terms of oxidative processes which may affect protein function in vivo.     

What is beta-carotene doing in the photosystem II reaction centre?

During photosynthesis carotenoids normally serve as antenna pigments, transferring singlet excitation energy to chlorophyll, and preventing singlet oxygen production from chlorophyll triplet states, by rapid spin exchange and decay of the carotenoid triplet to the ground state. The presence of two beta-carotene molecules in the photosystem II reaction centre (RC) now seems well established, but they do not quench the triplet state of the primary electron-donor chlorophylls, which are known as P(680). The beta-carotenes cannot be close enough to P(680) for triplet quenching because that would also allow extremely fast electron transfer from beta-carotene to P(+)(680), preventing the oxidation of water. Their transfer of excitation energy to chlorophyll, though not very efficient, indicates close proximity to the chlorophylls ligated by histidine 118 towards the periphery of the two main RC polypeptides. The primary function of the beta-carotenes is probably the quenching of singlet oxygen produced after charge recombination to the triplet state of P(680). Only when electron donation from water is disturbed does beta-carotene become oxidized. One beta-carotene can mediate cyclic electron transfer via cytochrome b559. The other is probably destroyed upon oxidation, which might trigger a breakdown of the polypeptide that binds the cofactors that carry out charge separation.     

A 1:2 crystalline complex of ApA:proflavine: a model for binding to single-stranded regions in RNA.

The structure of a 1"2 complex of adenylyl-(3',5')-adenosine phosphate and proflavine hemisulfate has been determined using the methods of x-ray crystallography. Since the ApA does not form a mini double helix, it may serve as a model for the interaction of planar molecules with single stranded nucleic acids. The dinucleotide adopts an extended conformation with the adenines in adjacent molecules forming base pairs. A most unusual feature of the molecule is that it does not obey the "rigid nucleotide" concept although none of the torsion angles occur in energetically unfavourable regions. This is most probably due to the strong interactions between the proflavine and the oligonucleotide.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5-4.0 mus duration dye laser flash of varying intensities as an excitation source. The yield (phi) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectoscopy at 515 nm, while the yield phi following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10-12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from phi utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (greater than 10(16) photon . cm-2) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1-2 mus after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules. At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse. A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a single-triplet bimolecular rate constant of gammast = 10(-8) cm3 . s-1, the following observations can be accounted for: (1) the rapid initial drop in phi and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Thermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-43

TDP-43 is a vital nucleic acid binding protein which forms stress-induced aberrant aggregates in around 97% cases of ALS, a fatal neurodegenerative disease. The functional tandem RRM domain of the protein (TDP-43^tRRM) has been shown to undergo amyloid-like aggregation under stress in a pH-dependent fashion. However, the underlying thermodynamic and molecular basis of aggregation and how the energy landscape of folding, stability, and aggregation are coupled and modulated by nucleic acid binding is poorly understood. Here, we show that the pH stress thermodynamically destabilizes the native protein and systematically populates the unfolded-like aggregation-prone molecules which leads to amyloid-like aggregation. We observed that specific DNA binding inhibits aggregation and populates native-like compact monomeric state even under low-pH stress as measured by circular dichroism, ANS binding, size exclusion chromatography, and transmission electron microscopy. We show that DNA-binding thermodynamically stabilizes and populates the native state even under stress and reduces the population of unfolded-like aggregation-prone molecules which leads to systematic aggregation inhibition. Our results suggest that thermodynamic modulation of the folding and aggregation energy landscape by nucleic-acid-like molecules could be a promising approach for effective therapeutic intervention in TDP-43-associated proteinopathies.     

Photophysical properties of quinones and their interaction with the photosynthetic reaction centre

Photophysical properties of tetramethyl-1,4-benzoquinone (TMBQ) and 2,6-dimethoxy-1,4-benzoquinone (DMOBQ) in solution and their interactions with the photosynthetic reaction centre (RC) isolated from the photosynthetic bacterium Rhodobacter sphaeroides have been investigated in this work. For these two benzoquinone derivatives an efficient ISC process which leads to the population of the lowest triplet state of the molecules upon direct excitation was observed. The presence of RC does not alter the properties of the triplet state of DMOBQ suggesting that interactions are negligible; on the other side RC efficiently quenched the T1 state of TMBQ. The behavior is rationalized in terms of redox potentials of quinones and kinetic characteristics of their transients.