Photodynamic inactivation of bacteria: Why it is not enough to excite a photosensitizer.

BACKGROUND: The development of multidrug resistance (MDR) in infectious agents is one of the most serious global problems facing humanity. Antimicrobial photodynamic therapy (APDT) shows encouraging results in the fight against MDR pathogens, including those in biofilms. METHODS: Photosensitizers (PS), monocationic methylene blue, polycationic and polyanionic derivatives of phthalocyanines, electroneutral and polycationic derivatives of bacteriochlorin were used to study photodynamic inactivation of Gram-positive and Gram-negative planktonic bacteria and biofilms under LED irradiation. Zeta potential measurements, confocal fluorescence imaging, and coarse-grained modeling were used to evaluate the interactions of PS with bacteria. PS aggregation and photobleaching were studied using absorption and fluorescence spectroscopy. RESULTS: The main approaches to ensure high efficiency of bacteria photosensitization are analyzed. CONCLUSIONS: PS must maintain a delicate balance between binding to exocellular and external structures of bacterial cells and penetration through the cell wall so as not to get stuck on the way to photooxidation-sensitive structures of the bacterial cell.

Mathematical Simulation of Electron Transport in the Primary Photosynthetic Processes.

Summarized results of investigation of regulation of electron transport and associated processes in the photosynthetic membrane using methods of mathematical and computer modeling carried out at the Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, are presented in this review. Detailed kinetic models of processes in the thylakoid membrane were developed using the apparatus of differential equations. Fitting of the model curves to the data of spectral measurements allowed us to estimate the values of parameters that were not determined directly in experiments. The probabilistic method of agent-based Monte Carlo modeling provides ample opportunities for studying dynamics of heterogeneous systems based on the rules for the behavior of individual elements of the system. Algorithms for simplified representation of Big Data make it possible to monitor changes in the photosynthetic apparatus in the course of culture growth in a photobioreactor and for the purpose of environmental monitoring. Brownian and molecular models describe movement and interaction of individual electron carrier proteins and make it possible to study electrostatic, hydrophobic, and other interactions leading to regulation of conformational changes in the reaction complexes. Direct multiparticle models explicitly simulate Brownian diffusion of the mobile protein carriers and their electrostatic interactions with multienzyme complexes both in solution and in heterogeneous interior of a biomembrane. The combined use of methods of kinetic and Brownian multiparticle and molecular modeling makes it possible to study the mechanisms of regulation of an integral system of electron transport processes in plants and algae at molecular and subcellular levels.

Molecular, Brownian, kinetic and stochastic models of the processes in photosynthetic membrane of green plants and microalgae.

The paper presents the results of recent work at the Department of Biophysics of the Biological Faculty, Lomonosov Moscow State University on the kinetic and multiparticle modeling of processes in the photosynthetic membrane. The detailed kinetic models and the rule-based kinetic Monte Carlo models allow to reproduce the fluorescence induction curves and redox transformations of the photoactive pigment P700 in the time range from 100 ns to dozens of seconds and make it possible to reveal the role of individual carriers in their formation for different types of photosynthetic organisms under different illumination regimes, in the presence of inhibitors, under stress conditions. The fitting of the model curves to the experimental data quantifies the reaction rate constants that cannot be directly measured experimentally, including the non-radiative thermal relaxation reactions. We use the direct multiparticle models to explicitly describe the interactions of mobile photosynthetic carrier proteins with multienzyme complexes both in solution and in the biomembrane interior. An analysis of these models reveals the role of diffusion and electrostatic factors in the regulation of electron transport, the influence of ionic strength and pH of the cellular environment on the rate of electron transport reactions between carrier proteins. To describe the conformational intramolecular processes of formation of the final complex, in which the actual electron transfer occurs, we use the methods of molecular dynamics. The results obtained using kinetic and molecular models supplement our knowledge of the mechanisms of organization of the photosynthetic electron transport processes at the cellular and molecular levels.

Explicit measurement of the endotoxin adsorption efficiency detects non-Langmuir behavior at low concentrations.

Lipopolysaccharides (LPS) are the Gram-negative bacteria cell wall components capable to induce the system inflammatory response even at picomolar concentrations. LPS detection at these concentrations is necessary to develop new sorbents for the efficient purification of the biological fluids. LAL-test widely used for LPS concentration estimation is based on the LPS biological activity measurement and thus may depend on the LPS concentration in a non-linear way. Here we propose a new explicit method for the LPS concentration measurement based on fluorescently labeled LPS and direct photon counting and develop the new protocol for LPS adsorption efficiency measurement. Following the suggested protocol in the experiments on novel sorbents, we demonstrate that LPS adsorption at small biologically relevant concentrations is non-Langmuir.

Mechanical properties of tubulin intra- and inter-dimer interfaces and their implications for microtubule dynamic instability.

Thirteen tubulin protofilaments, made of αß-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of ß-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αß-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.

Comparative analysis of plastocyanin-cytochrome f complex formation in higher plants, green algae and cyanobacteria.

Mechanisms of the complex formation between plastocyanin and cytochrome f in higher plants (Spinacia oleracea and Brassica rapa), green microalgae Chlamydomonas reinhardtii and two species of cyanobacteria (Phormidium laminosum and Nostoc sp.) were investigated using combined Brownian and molecular dynamics simulations and hierarchical cluster analysis. In higher plants and green algae, electrostatic interactions force plastocyanin molecule close to the heme of cytochrome f. In the subsequent rotation of plastocyanin molecule around the point of electrostatic contact in the vicinity of cytochrome f, copper (Cu) atom approaches cytochrome heme forming a stable configuration where cytochrome f molecule behaves as a rather rigid body without conformational changes. In Nostoc plastocyanin molecule approaches cytochrome f in a different orientation (head-on) where the stabilization of the plastocyanin-cytochrome f complex is accompanied by the conformational changes of the G188E189D190 loop that stabilizes the whole complex. In cyanobacterium P. laminosum, electrostatic preorientation of the approaching molecules was not detected, thus indicating that random motions rather than long-range electrostatic interactions are responsible for the proper mutual orientation. We demonstrated that despite the structural similarity of the investigated electron transport proteins in different photosynthetic organisms, the complexity of molecular mechanisms of the complex formation increases in the following sequence: non-heterocystous cyanobacteria - heterocystous cyanobacteria - green algae - flowering plants.

Molecular Mechanism of Uptake of Cationic Photoantimicrobial Phthalocyanine across Bacterial Membranes Revealed by Molecular Dynamics Simulations.

Phthalocyanines are aromatic macrocyclic compounds, which are structurally related to porphyrins. In clinical practice, phthalocyanines are used in fluorescence imaging and photodynamic therapy of cancer and noncancer lesions. Certain forms of the substituted polycationic metallophthalocyanines have been previously shown to be active in photodynamic inactivation of both Gram-negative and Gram-positive bacteria; one of them is zinc octakis(cholinyl)phthalocyanine (ZnPcChol8+). However, the molecular details of how these compounds translocate across bacterial membranes still remain unclear. In the present work, we have developed a coarse-grained (CG) molecular model of ZnPcChol8+ within the framework of the popular MARTINI CG force field. The obtained model was used to probe the solvation behavior of phthalocyanine molecules, which agreed with experimental results. Subsequently, it was used to investigate the molecular details of interactions between phthalocyanines and membranes of various compositions. The results demonstrate that ZnPcChol8+ has high affinity to both the inner and the outer model membranes of Gram-negative bacteria, although this species does not show noticeable affinity to the 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine membrane. Furthermore, we found out that the process of ZnPcChol8+ penetration toward the center of the outer bacterial membrane is energetically favorable and leads to its overall disturbance and formation of the aqueous pore. Such intramembrane localization of ZnPcChol8+ suggests their twofold cytotoxic effect on bacterial cells: (1) via induction of lipid peroxidation by enhanced production of reactive oxygen species (i.e., photodynamic toxicity); (2) via rendering the bacterial membrane more permeable for additional Pc molecules as well as other compounds. We also found that the kinetics of penetration depends on the presence of phospholipid defects in the lipopolysaccharide leaflet of the outer membrane and the type of counterions, which stabilize it. Thus, the results of our simulations provide a detailed molecular view of ZnPcChol8+ "self-promoted uptake", the pathway previously proposed for some small molecules crossing the outer bacterial membrane.

Multiparticle Brownian dynamics simulation of experimental kinetics of cytochrome bf oxidation and photosystem I reduction by plastocyanin.

A model of electron transport from cytochrome f to photosystem I mediated by plastocyanin was designed on the basis of the multiparticle Brownian dynamics method. The model combines events which occur over a wide time range, including protein diffusion along the thylakoid membrane, long-distance interactions between proteins, formation of a multiprotein complex, electron transfer within a complex and complex dissociation. Results of the modeling were compared with the experimental kinetics measured in chloroplast thylakoids. Computer simulation demonstrated that the complex interior of the photosynthetic membrane, electrostatic interactions and Brownian diffusion provide physical conditions for the directed electron flow along the photosynthetic electron transport chain.

Photosynthesis-related quantities for education and modeling.

A quantitative understanding of the photosynthetic machinery depends largely on quantities, such as concentrations, sizes, absorption wavelengths, redox potentials, and rate constants. The present contribution is a collection of numbers and quantities related mainly to photosynthesis in higher plants. All numbers are taken directly from a literature or database source and the corresponding reference is provided. The numerical values, presented in this paper, provide ranges of values, obtained in specific experiments for specific organisms. However, the presented numbers can be useful for understanding the principles of structure and function of photosynthetic machinery and for guidance of future research.

Computer simulation of interaction of photosystem 1 with plastocyanin and ferredoxin.

We designed 3D multiparticle computer models to simulate diffusion and interactions of spinach plastocyanin and ferredoxin with plant photosystem 1 in a solution. Using these models we studied kinetic characteristics of plastocyanin-photosystem 1 and ferredoxin-photosystem 1 complex formation at a variety of ionic strength values. The computer multiparticle models demonstrate non-monotonic dependences of complex formation rates on the ionic strength as the result of long-range electrostatic interactions. Our calculations show that the decrease in the association second-order rate constant at low values of the ionic strength is caused by the protein pairs spending more time in "wrong" orientations which do not satisfy the docking conditions and so do not form the final complex capable of the electron transfer.

New direct dynamic models of protein interactions coupled to photosynthetic electron transport reactions.

This review covers the methods of computer simulation of protein interactions taking part in photosynthetic electron transport reactions. A direct multiparticle simulation method that simulates reactions describing interactions of ensembles of molecules in the heterogeneous interior of a cell is developed. In the models, protein molecules move according to the laws of Brownian dynamics, mutually orient themselves in the electrical field, and form complexes in the 3D scene. The method allows us to visualize the processes of molecule interactions and to calculate the rate constants for protein complex formation reactions in the solution and in the photosynthetic membrane. Three-dimensional multiparticle computer models for simulating the complex formation kinetics for plastocyanin with photosystem I and cytochrome bf complex, and ferredoxin with photosystem I and ferredoxin:NADP+-reductase are considered. Effects of ionic strength are featured for wild type and mutant proteins. The computer multiparticle models describe nonmonotonic dependences of complex formation rates on the ionic strength as the result of long-range electrostatic interactions.