First biphotochromic fluorescent protein moxSAASoti stabilized for oxidizing environment.

Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of "mox"-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pKa values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pKa value is the C-O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C-O bond lengths are found for proteins with larger pKa. This feature can be utilized for prediction of the pKa values of red fluorescent proteins.

The role of cysteine residues in the allosteric modulation of the chromophore phototransformations of biphotochromic fluorescent protein SAASoti.

Biphotochromic fluorescent protein SAASoti contains five cysteine residues in its sequence and a V127T point mutation transforms it to the monomeric form, mSAASoti. These cysteine residues are located far from the chromophore and might control its properties only allosterically. The influence of individual, double and triple cysteine substitutions of mSAASoti on fluorescent parameters and phototransformation reactions (irreversible green-to-red photoconversion and reversible photoswitching) is studied. A set of mSAASoti mutant forms (C21N, C117S, C71V, C105V, C175A, C21N/C71V, C21N/C175A, C21N/C71G/C175A) is obtained by site-directed mutagenesis. We demonstrate that the C21N variant exists in a monomeric form up to high concentrations, the C71V substitution accelerates photoconversion to the red form and the C105V variant has the maximum photoswitching rate. All C175A-containing variants demonstrate different photoswitching kinetics and decreased photostability during subsequent switching cycles compared with other considered systems. Classical molecular dynamic simulations reveal that the F177 side chain located in the vicinity of the chromophore is considerably more flexible in the mSAASoti compared with its C175A variant. This might be the explanation of the experimentally observed slowdown the thermal relaxation rate, i.e., trans-cis isomerization of the chromophore in mSAASoti upon C175A substitution.

Sensors for Proteolytic Activity Visualization and Their Application in Animal Models of Human Diseases.

Various sensors designed for optical and photo(opto)acoustic imaging in living systems are becoming essential components of basic and applied biomedical research. Some of them including those developed for determining enzyme activity in vivo are becoming commercially available. These sensors can be used for various fluorescent signal detection methods: from whole body tomography to endoscopy with miniature cameras. Sensor molecules including enzyme-cleavable macromolecules carrying multiple quenched near-infrared fluorophores are able to deliver their payload in vivo and have long circulation time in bloodstream enabling detection of enzyme activity for extended periods of time at low doses of these sensors. In the future, more effective "activated" probes are expected to become available with optimized sensitivity to enzymatic activity, spectral characteristics suitable for intraoperative imaging of surgical field, biocompatibility and lack of immunogenicity and toxicity. New in vivo optical imaging methods such as the fluorescence lifetime and photo(opto)acoustic imaging will contribute to early diagnosis of human diseases. The use of sensors for in vivo optical imaging will include more extensive preclinical applications of experimental therapies. At the same time, the ongoing development and improvement of optical signal detectors as well as the availability of biologically inert and highly specific fluorescent probes will further contribute to the introduction of fluorescence imaging into the clinic.

Magnetization and ferromagnetic resonance in a Fe/Gd multilayer: experiment and modelling.

Static and dynamic magnetic properties of a [Fe(35 Å)/Gd(50 Å)]12 superlattice are investigated experimentally in the temperature range 5-295 K using SQUID magnetometery and the ferromagnetic resonance (FMR) technique at frequencies 7-38 GHz. The obtained magnetization curves and FMR spectra are analysed theoretically using numerical simulation on the basis of the effective field model. At every given temperature, both static and resonance experimental data can be approximated well within the proposed model. However, a considerable temperature dependence of the effective field parameter in gadolinium layers has to be taken into account to achieve reasonable agreement with the experimental data in the entire temperature range studied. To describe the peculiarities of experimental FMR spectra, a non-local diffusion-type absorption term in Landau-Lifshitz equations is considered in addition to the Gilbert damping term. Possible reasons for the observed effects are discussed.

Hydrogen bonding of nitroxide spin labels in membrane proteins.

On the basis of experiments at 275 GHz, we reconsider the dependence of the continuous-wave EPR spectra of nitroxide spin-labeled protein sites in sensory- and bacteriorhodopsin on the micro-environment. The high magnetic field provides the resolution necessary to disentangle the effects of hydrogen bonding and polarity. In the gxx region of the 275 GHz EPR spectrum, bands are resolved that derive from spin-label populations carrying no, one or two hydrogen bonds. The gxx value of each population varies hardly from site to site, significantly less than deduced previously from studies at lower microwave frequencies. The fractions of the populations vary strongly, which provides a consistent description of the variation of the average gxx and the average nitrogen-hyperfine interaction Azz from site to site. These variations reflect the difference in the proticity of the micro-environment, and differences in polarity contribute marginally. Concomitant W-band ELDOR-detected NMR experiments on the corresponding nitroxide in perdeuterated water resolve population-specific nitrogen-hyperfine bands, which underlies the interpretation for the proteins.

Structural and dynamical characteristics of trehalose and sucrose matrices at different hydration levels as probed by FTIR and high-field EPR.

Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, particularly trehalose and sucrose. Trehalose is most effective also in protecting isolated in vitro biostructures. In an attempt to clarify the molecular mechanisms of disaccharide bioprotection, we compared the structure and dynamics of sucrose and trehalose matrices at different hydration levels by means of high-field W-band EPR and FTIR spectroscopy. The hydration state of the samples was characterized by FTIR spectroscopy and the structural organization was probed by EPR using a nitroxide radical dissolved in the respective matrices. Analysis of the EPR spectra showed that the structure and dynamics of the dehydrated matrices as well as their evolution upon re-hydration differ substantially between trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms of distribution of the residual water and spin-probe molecules. In contrast, dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose polycrystalline clusters and several bulk water domains. The amorphous form was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a structural homogenization of the sucrose matrix, whilst in the trehalose matrix several domains develop differing in the local water/radical content and radical mobility. The molecular model of the matrices provides an explanation for the different protein-matrix dynamical coupling observed in dried ternary sucrose and trehalose matrices, and accounts for the superior efficacy of trehalose as a bioprotectant. Furthermore, for bacterial photosynthetic reaction centers it is shown that at low water content the protein-matrix coupling is modulated by the sugar/protein molar ratio in sucrose matrices only. This effect is suggested to be related to the preference for sucrose, rather than trehalose, as a bioprotective disaccharide in some anhydrobiotic organisms.

Orientation resolving dipolar high-field EPR spectroscopy on disordered solids: II. Structure of spin-correlated radical pairs in photosystem I.

The distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on electron transfer proteins, we report the results of orientation resolving dipolar high-field EPR spectroscopy on the charge-separated state P700•+ A1•­ (P700, primary electron donor; A1, phylloquinone electron acceptor) in Photosystem I (PS I). Pulsed high-field EPR spectroscopy at W-band (95 GHz, 3.4 T) with extensions to PELDOR (pulsed electron­electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) was utilized to obtain the parameters describing the three-dimensional structure of the laser-flash-induced transient radical pair P700•+ A1•­ in a frozen solution of deuterated PS I from the cyanobacterium Synechocystis sp. PCC 6803, which is performing oxygenic photosynthesis. The measured distances and relative orientations of the weakly coupled radical ions in the radical pair P700•+ A1•­ are compared with previously reported geometries and with those of the precursor cofactors P700 and A1 known from X-ray crystallography. Cyclic electron transfer was found to proceed exclusively via the A-branch of the cofactor chain of PS I at cryogenic temperature. The position and orientation of the reduced phylloquinone coincide with those of the precursor, revealing that no substantial orientational changes of the phylloquinone molecule upon charge separation occur. Several distinct orientations of the P700•+ g-tensor axes with respect to the molecular frame of the primary donor were found experimentally, which we explain by several conformational substates of the P700•+ radical structure having slightly different electron spin density distributions.

An improved coupling design for high-frequency TE011 electron paramagnetic resonance cavities.

In high-frequency electron paramagnetic resonance (EPR) spectroscopy the sample is usually accommodated in a single-mode cylindrical TE(011) microwave cavity. This cavity stands out in terms of flexibility for various types of EPR experiments due to convenient control of its resonance frequency and easy waveguide-to-cavity microwave coupling. In continuous wave and in pulsed EPR it is, however, essential to be able to vary the coupling efficiency over a large range. We present a new mechanical design to vary the microwave coupling to the cavity using a movable metal sphere. This coupling sphere is shifted in the plane of the iris wall inside the coupling waveguide. The design allows for a compact and robust construction of the EPR probehead that can be easily accommodated inside a limited space of helium flow cryostat. The construction details and characterization of the coupling element for 95 GHz (W-band) EPR as well as for 34 GHz (Q-band) are presented.

Biodistribution and stability of CdSe core quantum dots in mouse digestive tract following per os administration: advantages of double polymer/silica coated nanocrystals.

CdSe-core, ZnS-capped semiconductor quantum dots (QDs) are of great potential for biomedical applications. However, applications in the gastrointestinal tract for in vivo imaging and therapeutic purposes are hampered by their sensitivity to acidic environments and potential toxicity. Here we report the use of coatings with a combination of polythiol ligands and silica shell (QDs PolyT-APS) to stabilize QDs fluorescence under acidic conditions. We demonstrated the stability of water-soluble QDs PolyT-APS both in vitro, in strong acidic solutions, and in vivo. The biodistribution, stability and photoluminescence properties of QDs in the gastrointestinal tract of mice after per os administration were assessed. We demonstrated that QDs coated with current traditional materials - mercapto compounds (QDs MPA) and pendant thiol group (QDs PolyT) - are not capable of protecting QDs from chemically induced degradation and surface modification. Polythiol ligands and silica shell quantum dots (QDs PolyT-APS) are suitable for biological and biomedical applications in the gastrointestinal tract.

Using lanthanide-based resonance energy transfer for in vitro and in vivo studies of biological processes.

This review describes key directions in the development of different probes based on complex compounds of lanthanides for in vitro and in vivo researches. The role of microsecond fluorescence of lanthanides for overcoming problems of background fluorescence is considered. The basic classes of synthetic and genetically encoded complex compounds of lanthanides are summarized. Main principles of designing lanthanide-based molecular sensors, including FRET sensors based on lanthanides and colored fluorescent proteins are described. Their applications in bioanalytical research and cellular bioimaging are described. The main principles of cellular bioimaging using lanthanides are formulated, questions of their delivery into cells are considered, and the problem of their potential toxicity for living organisms is discussed. A technique using multiphoton excitation of lanthanides is described.

Computational strategy for tuning spectral properties of red fluorescent proteins.

Computational methods of quantum chemistry are used to characterize structures and vertical excitation energies of the S(0)-S(1) optical transitions in the chromophore binding pockets of the red fluorescent proteins DsRed and of its artificial mutant mCherry. As previously shown, optimizing the equilibrium geometry configurations with B3LYP density functional theory, followed by ZINDO calculations of the electronic excitations, yields positions of the optical bands in good agreement with experimental data. These large scale quantum calculations elucidate the role of the hydrogen bonded network as well as point mutations in the absorption spectra of the DsRed and mCherry proteins. The effect of an external electric field applied to the fluorescent protein chromophores is examined and shows that such fields may result in large shifts in spectral bands. These strategies can be applied for rational design of the fluorescent proteins by site-directed mutagenesis.

B-branch electron transfer in the photosynthetic reaction center of a Rhodobacter sphaeroides quadruple mutant. Q- and W-band electron paramagnetic resonance studies of triplet and radical-pair cofactor states.

The directionality of light-induced charge transfer in bacterial photosynthetic reaction centers (RCs) with respect to their A and B cofactor branches is still poorly understood on the electronic level. A prominent example is primary electron transfer in the RCs from the purple bacterium Rb. sphaeroides. Site-directed mutants with specific alterations of the cofactor binding sites with respect to the native system can deliver useful information toward a better understanding of the directionality enigma. Here we report on electron paramagnetic resonance (EPR) studies of the LDHW quadruple mutant, HL(M182)/GD(M203)/LH(M214)/AW(M260), which contains crucial mutations in the electron-transfer pathway. The directionality of the charge separation process was studied under light- or dark-freezing conditions first directly by 95 GHz (W-band) high-field EPR spectroscopy examining the charge-separated radical pairs (P865•+ Q(B)•−) of the primary donor P865, a bacteriochlorophyll dimer, and the terminal acceptor, QB, a ubiquinone-10. Second, it was studied indirectly by 34 GHz (Q-band) EPR examining the triplet states of the primary donor ((3)P865) that occur as a byproduct of the photoreaction. At 10 K, the triplet state has been found to derive mainly from an intersystem crossing mechanism, indicating the absence of charge-separated radical-pair states with a lifetime longer than 10 ns. B-branch charge separation and formation of the triplet-state (3)P865 via a radical-pair mechanism can be induced with low yield at 10 K by direct excitation of the bacteriopheophytins in the B-branch at 537 nm. At this wavelength, charge separation most probably proceeds via hole transfer from bacteriopheophytin to the primary donor. The triplet state of the primary donor is found to be quenched by the carotenoid cofactor present in the RC. The light-induced transient EPR signal of P•+ Q(B)•− is formed in a minor fraction of RCs (<1%) for RCs frozen in the dark. In contrast, about 70% of RCs illuminated upon freezing are trapped in the long-lived (τ > 104 s) charge-separated-state P•+ Q(B)•−. The temperature dependence of the EPR signals from P•+ Q(B)•− points to two factors responsible for the forward electron transfer to the terminal acceptor QB and for the charge-recombination reaction. The first factor involves a significant protein conformational change to initiate P•+ Q(B)•− charge separation, presumably by moving the quinone from the distal to the proximal position relative to the iron. The second factor includes protein relaxation, which governs the charge-recombination process along the B-branch pathway of the LDHW mutant.

Computer modeling of the structure and spectra of fluorescent proteins.

Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers.

High-field EPR and ESEEM investigation of the nitrogen quadrupole interaction of nitroxide spin labels in disordered solids: toward differentiation between polarity and proticity matrix effects on protein function.

The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.

Conformation dependence of pKa's of the chromophores from the purple asFP595 and yellow zFP538 fluorescent proteins.

Two members of the green fluorescent protein family, the purple asFP595 and yellow zFP538 proteins, are perspective fluorescent markers for use in multicolor imaging and resonance energy-transfer applications. We report the results of quantum based calculations of the solution pKa values for selected protonation sites of the denatured asFP595 and zFP538 chromophores in the trans- and cis-conformations in order to add in the interpretation of photophysical properties of these proteins. The pKa values were determined from the theromodynamic cycle based on B3LYP/6-311++G(2df,2p) calculations of the gas phase free energies of the molecules and the B3LYP/6-311++G(d,p) calculations of solvation energies. The results show that the pKa's of the protonation sites of the chromophore from asFP595 noticeably depend on the isomer conformation (cis- or trans-), while those of zFP538 are much less sensitive to isomerization.

Orientation-resolving pulsed electron dipolar high-field EPR spectroscopy on disordered solids: I. Structure of spin-correlated radical pairs in bacterial photosynthetic reaction centers.

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action.

In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

High-field EPR spectroscopy applied to biological systems: characterization of molecular switches for electron and ion transfer.

The last decade witnessed a tremendous growth in combined efforts of biologists, chemists and physicists to understand the dominant factors determining the specificity and directionality of transmembrane transfer processes in proteins. A large variety of experimental techniques is being used including X-ray and neutron diffraction, but also time-resolved optical, infrared and magnetic resonance spectroscopy. This is done in conjunction with genetic engineering strategies to construct site-specific mutants for controlled modification of the proteins. As a general perception of these efforts, the substantial influence of weak interactions within the protein and its membrane interfaces is recognized. The weak interactions are subject to subtle changes during the reaction cycle owing to the inherent flexibility of the protein-membrane complex. Specific conformational changes accomplish molecular-switch functions for the transfer process to proceed with optimum efficiency. Characteristic examples of time varying non-bonded interactions are specific H-patterns and/or polarity effects of the microenvironment. The present perception has emerged from the coupling of newly developed spectroscopic techniques - and advanced EPR certainly deserves credit in this respect - with newly developed computational strategies to interpret the experimental data in terms of protein structure and dynamics. By now, the partners of this coupling, particularly high-field EPR spectroscopy and DFT-based quantum theory, have reached a level of sophistication that applications to large biocomplexes are within reach. In this review, a few large paradigm biosystems are surveyed which were explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three biosystems are characterized with respect to structure and dynamics: (1) Light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from the photosynthetic bacterium Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The detailed information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide highly desired detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales. The review introduces the audience to origins and basic experiments of EPR in relation to NMR, describes the underlying strategies for extending conventional EPR to high-field/high-frequency EPR, and highlights those details of molecular information that are obtained from high-field EPR in conjunction with genetic engineering and that are not accessible by "classical" spectroscopy. The importance of quantum-chemical interpretation of the experimental data by DFT and advanced semiempirical molecular-orbital theory is emphasized. A short description of the laboratory-built 95 GHz and 360 GHz EPR/ENDOR spectrometers at FU Berlin is also presented. The review concludes with an outlook to future opportunities and challenges of advanced bio-EPR in interdisciplinary research.

Hydrotechnical facilities within the Chernobyl nuclear power plant exclusion zone: impacts on hydrologic regime and plant growth patterns of floodplain water bodies of the Pripyat River.

As result of the Chernobyl nuclear power plant accident the territory of the left-bank flood-lands of the Pripyat River have undergone intensive radionuclide contamination. With the purpose of preventing the washing away of radioactive substances, a complex of flood protection dams was constructed. This construction changed the hydrological regime of these territories and caused overgrowth by higher aquatic plants. Absence of a flowing mode of reservoirs, the stagnant phenomena during spring and seasonal high waters on the embank site have caused amplification of eutrophication processes, swamping and, connected with it, increase of water-marsh floristic complex in the structure of the vegetative cover.

Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence.

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.