Showing papers in "Biophysics in 2016"

Molecular dynamics analysis to evaluate docking pose prediction

Abstract: The accurate prediction of a ligand-protein complex structure is important for computer-assisted drug development. Although many docking methods have been developed over the last three decades, the success of binding structure prediction remains greatly limited. The purpose of this study was to demonstrate the usefulness of molecular dynamics (MD) simulation in assessing a docking pose predicted using a docking program. If the predicted pose is not unstable in an aqueous environment, MD simulation equilibrates the system and removes the ligand from the predicted position. Here we investigated two proteins that are important potential therapeutic targets: β2 adrenergic receptor (β2AR) and PR-Set7. While β2AR is rigid and its ligands are very similar to the template ligand (carazolol), PR-Set7 is very flexible and its ligands vary greatly from the template ligand (histone H4 tail peptide). On an empirical basis, we usually expect that the docking prediction is accurate when the protein is rigid and its ligands are similar to the template ligand. The MD analyses in this study clearly suggested such a tendency. Furthermore, we discuss the possibility that the MD simulation can predict the binding pose of a ligand.

Mass spectrometric analysis of protein–ligand interactions

Abstract: The interactions of small molecules with proteins (protein-ligand interactions) mediate various biological phenomena including signal transduction and protein transcription and translation. Synthetic compounds such as drugs can also bind to target proteins, leading to the inhibition of protein-ligand interactions. These interactions typically accompany association-dissociation equilibrium according to the free energy difference between free and bound states; therefore, the quantitative biophysical analysis of the interactions, which uncovers the stoichiometry and dissociation constant, is important for understanding biological reactions as well as for rational drug development. Mass spectrometry (MS) has been used to determine the precise molecular masses of molecules. Recent advancements in MS enable us to determine the molecular masses of protein-ligand complexes without disrupting the non-covalent interactions through the gentle desolvation of the complexes by increasing the vacuum pressure of a chamber in a mass spectrometer. This method is called MS under non-denaturing conditions or native MS and allows the unambiguous determination of protein-ligand interactions. Under a few assumptions, MS has also been applied to determine the dissociation constants for protein-ligand interactions. The structural information of a protein-ligand interaction, such as the location of the interaction and conformational change in a protein, can also be analyzed using hydrogen/deuterium exchange MS. In this paper, we briefly describe the history, principle, and recent applications of MS for the study of protein-ligand interactions.

Electrical signals in higher plants: Mechanisms of generation and propagation

Abstract: Local stimulation induces generation and propagation of electric signals in higher plants. Noninvasive stimulus induces an action potential and damaging influences lead to the variation potential. The mechanism of the generation of an action potential is rather complex in nature and is associated with both activation of ion channels (Ca2+, Cl–, and K+) and transient change in the activity of the plasma membrane H+-ATPase. Generation of the variation potential, the duration of which is considerably longer than that of the action potential, is based on transient inactivation of the electrogenic pump; however, passive ion fluxes also contribute to such process, which causes qualitative similarity of the mechanisms of action potential and variation potential generation. Propagation of electrical signals mainly occurs in conducting bundles; thus, transfer of an action potential is associated with vascular parenchyma and sieve elements, while the variation potential is connected to the xylem vessels. The mechanism of the distribution the action potential is similar to nerve impulse transmission, while generation of the variation potential is induced by transfer of a chemical substance, whose propagation is accelerated by a hydraulic wave.

myPresto/omegagene: a GPU-accelerated molecular dynamics simulator tailored for enhanced conformational sampling methods with a non-Ewald electrostatic scheme.

Abstract: Molecular dynamics (MD) is a promising computational approach to investigate dynamical behavior of molecular systems at the atomic level. Here, we present a new MD simulation engine named "myPresto/omegagene" that is tailored for enhanced conformational sampling methods with a non-Ewald electrostatic potential scheme. Our enhanced conformational sampling methods, e.g., the virtual-system-coupled multi-canonical MD (V-McMD) method, replace a multi-process parallelized run with multiple independent runs to avoid inter-node communication overhead. In addition, adopting the non-Ewald-based zero-multipole summation method (ZMM) makes it possible to eliminate the Fourier space calculations altogether. The combination of these state-of-the-art techniques realizes efficient and accurate calculations of the conformational ensemble at an equilibrium state. By taking these advantages, myPresto/omegagene is specialized for the single process execution with Graphics Processing Unit (GPU). We performed benchmark simulations for the 20-mer peptide, Trp-cage, with explicit solvent. One of the most thermodynamically stable conformations generated by the V-McMD simulation is very similar to an experimentally solved native conformation. Furthermore, the computation speed is four-times faster than that of our previous simulation engine, myPresto/psygene-G. The new simulator, myPresto/omegagene, is freely available at the following URLs: http://www.protein.osaka-u.ac.jp/rcsfp/pi/omegagene/ and http://presto.protein.osaka-u.ac.jp/myPresto4/.

Primary physical mechanism of the biological effects of weak magnetic fields

Abstract: The primary physical mechanism of the magnetoreception of weak magnetic fields is considered. It imposes limits on the magnetic biological effect at the stage prior to the involvement of specific biophysical and biochemical mechanisms, i.e., regardless of the nature of the target of the magnetic field. It has been shown that the biological effects of weak magnetic fields have, in general, non-linear and spectral properties. Observation of these characteristics gives information not only on the gyromagnetic ratio, but also on the parameters of the interaction between the target and its immediate surroundings. This makes it possible for one to develop schemes for the identification of the biophysical mechanisms of magnetoreception.

Density functional study of molecular interactions in secondary structures of proteins.

Abstract: Proteins play diverse and vital roles in biology, which are dominated by their three-dimensional structures. The three-dimensional structure of a protein determines its functions and chemical properties. Protein secondary structures, including α-helices and β-sheets, are key components of the protein architecture. Molecular interactions, in particular hydrogen bonds, play significant roles in the formation of protein secondary structures. Precise and quantitative estimations of these interactions are required to understand the principles underlying the formation of three-dimensional protein structures. In the present study, we have investigated the molecular interactions in α-helices and β-sheets, using ab initio wave function-based methods, the Hartree-Fock method (HF) and the second-order Moller-Plesset perturbation theory (MP2), density functional theory, and molecular mechanics. The characteristic interactions essential for forming the secondary structures are discussed quantitatively.

In silico studies for the interaction of tumor necrosis factor-alpha (TNF-α) with different saponins from Vietnamese ginseng ( Panax vietnamesis ).

Abstract: Tumor necrosis factor-alpha (TNF-α) is a cytokine that plays an important role in inflammatory process and tumor development. Recent studies demonstrate that triterpene saponins from Vietnamese ginseng are efficient inhibitors of TNF-α. But the interactions between TNF-α and the saponins are still unclear. In this study, molecular docking and molecular dynamics simulations of TNF-α with three different triterpene saponins (majonoside R2, vina-ginsenoside R1 and vina-ginsenoside R2) were performed to evaluate their binding ability. Our results showed that the triterpene saponins have a good binding affinity with protein TNF-α. The saponins were docked to the pore at the top of the "bell" or "cone" shaped TNF-α trimer and the complexes were structurally stable during 100 ns molecular dynamics simulation. The predicted binding sites would help to subsequently investigate the inhibitory mechanism of triterpene saponins.

Forced Oscillations of DNA Bases

Abstract: This paper presents the results of a study of forced angular oscillations of DNA bases using a mathematical model that consists of two coupled nonlinear differential equations that take the effects of dissipation and the influence of an external periodic field into account. The calculation results are illustrated for the sequence of the gene that codes for interferon alpha 17 (IFNA17).

Accelerating in vitro studies on circadian clock systems using an automated sampling device.

Abstract: KaiC, a core protein of the cyanobacterial circadian clock, is rhythmically autophosphorylated and autodephosphorylated with a period of approximately 24 h in the presence of two other Kai proteins, KaiA and KaiB. In vitro experiments to investigate the KaiC phosphorylation cycle consume considerable time and effort. To automate the fractionation, quantification, and evaluation steps, we developed a suite consisting of an automated sampling device equipped with an 8-channel temperature controller and accompanying analysis software. Eight sample tables can be controlled independently at different temperatures within a fluctuation of ±0.01°C, enabling investigation of the temperature dependency of clock activities simultaneously in a single experiment. The suite includes an independent software that helps users intuitively conduct a densitometric analysis of gel images in a short time with improved reliability. Multiple lanes on a gel can be detected quasi-automatically through an auto-detection procedure implemented in the software, with or without correction for lane ‘smiling.’ To demonstrate the performance of the suite, robustness of the period against temperature variations was evaluated using 32 datasets of the KaiC phosphorylation cycle. By using the software, the time required for the analysis was reduced by approximately 65% relative to the conventional method, with reasonable reproducibility and quality. The suite is potentially applicable to other clock or clock-related systems in higher organisms, relieving users from having to repeat multiple manual sampling and analytical steps.

Thymoquinone, a biologically active component of Nigella sativa , induces mitochondrial production of reactive oxygen species and programmed death of tumor cells

Abstract: Mechanisms of tumor-cell responses to 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and 1,4-benzoquinone were studied using fluorescence and the inhibition assay. It was shown that quinones enhanced the intracellular production of reactive oxygen species, reduced the mitochondrial membrane potential, and induced tumor-cell death through different pathways. It was found that thymoquinone, which induced lower production of reactive oxygen species than 1,4-benzoquinone, was more toxic to tumor cells. It was established that reactive oxygen species produced due to exposure to thymoquinone are involved in redox signaling processes that lead to the formation of mitochondrial permeability transition pores and activation of programmed cell death. These results suggest that the functioning of the established redox signaling mechanism is enabled by the colocalization of mitochondrial oxidoreductases involved in the production of reactive oxygen species and of protein targets of reactive oxygen species involved in the activation of apoptosis.

The role of parvalbumin-containing interneurons in the regulation of spontaneous synchronous activity of brain neurons in culture

Abstract: Subtypes of inhibitory GABAergic neurons containing Ca2+-binding proteins play a pivotal role in the regulation of spontaneous synchronous [Ca2+] i transients in a neuronal network. In this study it is shown that: (1) the interneurons that containing Ca2+-binding proteins at buffer concentration can be identified by the shape of Ca2+-signa1 in response to depolarization or activation of ionotropic glutamate receptors; (2) Ca2+-binding proteins are involved in desynchronization of spontaneous Ca2+ transients. At low frequencies of spontaneous synchronous [Ca2+] i transients (less than 0.2 Hz) neurons show quasi-synchronous pulsations. At higher frequencies, synchronization of spontaneous synchronous [Ca2+] i transients occurs in all neurons; (3) it is established that several synchronous oscillations with different frequencies coexist in the network and the amplitude of their depolarizing pulse also varies. This phenomenon is apparently the mechanism that selectively directs information in separate neurons using the same network; and (4) in one population of interneurons at high frequencies of spontaneous synchronous [Ca2+] i transients the inversion of Cl– concentration gradient is observed. In this case, the inhibition of GABA(A) receptors suppresses the activity of neurons in this population and excites other neurons in the network. Thus, the GABAergic neurons that contain Ca-binding proteins show different mechanisms to regulate the synchronous neuronal activities in cultured rat hippocampal cells.

Metabolic pathway reconstruction strategies for central metabolism and natural product biosynthesis

Abstract: Metabolic pathway reconstruction presents a challenge for understanding metabolic pathways in organisms of interest. Different strategies, i.e., reference-based vs. de novo, must be used for pathway reconstruction depending on the availability of well-characterized enzymatic reactions. If at least one enzyme is already known to catalyze a reaction, its amino acid sequence can be used as a reference for identifying homologous enzymes in the genome of an organism of interest. Where there is no known enzyme able to catalyze a corresponding reaction, however, the reaction and the corresponding enzyme must be predicted de novo from chemical transformations of the putative substrate-product pair. This review summarizes studies involving reference-based and de novo metabolic pathway reconstruction and discusses the importance of the classification and structure-function relationships of enzymes.

Solution of Levinthal’s paradox is possible at the level of the formation and assembly of protein secondary structures

Abstract: The complete volume of a protein’s conformation space is smaller by many orders of magnitude at the level of secondary-structure elements as compared with the conformation of amino-acid residues. According to Levinthal’s estimate, the latter is ~102L, with L being the number of residues in the chain, while the former, at the level of secondary structures, increases no faster than ~LN with N being the number of the secondary-structure elements. N is approximately L/15 according to the statistics of protein structures. This drastic decrease in the exponent (L/15 instead of 2L) considerably reduces the sampling space and explains the reason that sampling of the conformation space at the level of secondary-structure elements does not prevent the protein chain from finding its most stable structure.

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains

Abstract: Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

Changes in the kinetics of plasma chemiluminescence as a measure of systemic oxidative stress in humans

Abstract: Oxidative stress acts as a pathogenetic factor in many diseases; estimating its level is important for early diagnosis and therapy adjustment. The antioxidant status was evaluated for the blood plasma. A set of chemiluminescence (CL) kinetic-curve parameters (latent period τlat and analytical signal increment ΔI CL) in a 2,2’-azo-bis(2-amidinopropane)dihydrochloride–luminol system were proposed for estimating the oxidative stress level. Uric acid and albumin were identified as major components that are responsible for the changes in the plasma CL kinetic curve. UV light caused oxidative modification of serum albumin in a dose-dependent manner, thus enhancing its antioxidant properties. Changes in plasma CL kinetics were proposed as a means to measure oxidative stress in the human body.

Relaxation folding and the principle of the minimum rate of energy dissipation for conformational motions in a viscous medium

Abstract: A numerical simulation of the folding of a model polymer chain of 50 units with valence bonds of a fixed length and fixed valence angle values has been performed using the strong friction approximation. The rate of energy dissipation in the system has been analyzed for conformational motions along a trajectory determined by the equations of mechanics and the trajectories characterized by random and variable deviations from the mechanical path. The validity of the principle of the minimum average rate of the energy dissipation for the conformational relaxation of a macromolecule in a viscous medium has been demonstrated. A profile of the relaxation energy funnel for the folding of a macromolecular chain has been constructed. Slow and rapid stages of folding could be distinguished in the energy funnel profile; the final state was separated from the nearest conformations of the folded chain by an energy gap.

Modeling of primary photosynthetic processes using the kinetic Monte Carlo method

Abstract: Processes that occur in the ensemble of photosynthetic electron transport systems have been modeled using a kinetic Monte Carlo method. The size of a simulated ensemble (3–5 million elementary photosynthetic chains) corresponds to the number of photosynthetic reaction centers in a plant cell. The method enables one to modify the structure of a model system according to different concepts of the organization of processes in a photosynthetic membrane. Using this model, the experimental kinetics of the chlorophyll fluorescence induction associated with the Photosystem II and the redox transformations of a photoactive pigment of the Photosystem I have been successfully reproduced. The model was verified by comparing the calculated fluorescence induction curves to experimental curves, obtained in the presence of various photosynthesis inhibitors and under temperature inactivation of the Photosystem II donor side.

The Effect of a “Zero” Magnetic Field on the Production of Reactive Oxygen Species in Neutrophils

Abstract: Exposure of mouse peritoneal neutrophils to hypomagnetic conditions (magnetic shielding, a residual static magnetic field of 20 nT) for 1.5 h decreased the level of intracellular reactive oxygen species as recorded by changes in the fluorescence intensity of 2,7-dichlorodihydrofluorescein and dihydrorhodamine 123 oxidation products. The effect of a hypomagnetic field was similarly observed after adding a respiratory burst activator (the formylated peptide N-formyl–Met–Leu–Phe or phorbol 12-meristate-13-acetate) to a low concentration.

Priming of the respiratory burst in neutrophils exposed to a combination of weak constant and alternating low-frequency magnetic fields in vitro

Abstract: An hour-long exposure of peritoneal neutrophils of mice to a combination of a weak constant magnetic field (42 μT) and low-frequency alternating magnetic fields collinear to the weak constant magnetic field (frequencies 1, 4.4, and 16.5 Hz, total amplitude 0.86 μT) at physiological temperatures promoted a significant increase in chemiluminescence of cells in response to subsequent exposure to low concentrations of respiratory burst activators (formylated peptide N-formyl-Met–Leu–Phe or phorbol ester phorbol-12-myristate-13-acetate) in the presence of luminol. The response of human neutrophils isolated from peripheral blood to the pretreatment with combined magnetic fields followed by exposure to the activator N-formyl-Met–Leu–Phe was similar to the response of mouse neutrophils.

The Possible Role of Nonbilayer Structures in Regulating ATP Synthase Activity in Mitochondrial Membranes.

Abstract: The effects of temperature and of the membrane-active protein CTII on the formation of nonbilayer structures in mitochondrial membranes were studied by 31P-NMR. Increasing the temperature of isolated mitochondrial fractions correlated with an increase in ATP synthase activity and the formation of nonbilayer packed phospholipids with immobilized molecular mobility. Computer modeling was employed for analyzing the interaction of mitochondrial membrane phospholipids with the molecular surface of CTII, which behaves like a dicyclohexylcarbodiimide-binding protein (DCCD-BP) of the F0 group in a lipid phase. Overall, our studies suggest that proton permeability toroidal pores formed in mitochondrial membranes consist of immobilized nonbilayer-packed phospholipids formed via interactions with DCCD-BP. Our studies support the existence of a proton transport along a concentration gradient mediated via transit toroidal permeability pores which induce conformational changes necessary for mediating the catalytic activity of ATP synthase in the subunits of the F0-F1 complex.

Cation-limited kinetic model for microbial extracellular electron transport via an outer membrane cytochrome C complex.

Abstract: Outer-membrane c-type cytochrome (OM c-Cyt) complexes in several genera of iron-reducing bacteria, such as Shewanella and Geobacter, are capable of transporting electrons from the cell interior to extracellular solids as a terminal step of anaerobic respiration. The kinetics of this electron transport has implications for controlling the rate of microbial electron transport during bioenergy or biochemical production, iron corrosion, and natural mineral cycling. Herein, we review the findings from in-vivo and in-vitro studies examining electron transport kinetics through single OM c-Cyt complexes in Shewanella oneidensis MR-1. In-vitro electron flux via a purified OM c-Cyt complex, comprised of MtrA, B, and C proteins from S. oneidensis MR-1, embedded in a proteoliposome system is reported to be 10- to 100-fold faster compared with in-vivo estimates based on measurements of electron flux per cell and OM c-Cyts density. As the proteoliposome system is estimated to have 10-fold higher cation flux via potassium channels than electrons, we speculate that the slower rate of electron-coupled cation transport across the OM is responsible for the significantly lower electron transport rate that is observed in-vivo. As most studies to date have primarily focused on the energetics or kinetics of interheme electron hopping in OM c-Cyts in this microbial electron transport mechanism, the proposed model involving cation transport provides new insight into the rate detemining step of EET, as well as the role of self-secreted flavin molecules bound to OM c-Cyt and proton management for energy conservation and production in S. oneidensis MR-1.

The biophysical aspects of photodynamic therapy

Abstract: The photodynamic effect, viz., photodamage of stained cells in the presence of oxygen, is used for destruction of tumors and other abnormal cells. The present review considers the biophysical mechanisms of the photodynamic action on cells. The importance of two major mechanisms of photodynamic damage of cells is discussed. The first one is mediated by electron or proton transfer, whereas the second one involves singlet oxygen. Another question that is considered is the importance of oxidation of membrane lipids or proteins for the photodynamic damage of cells. The phototransformation of photosensitizers and their intracellular localization and delivery to cells and tissues that have undergone abnormal changes are discussed. The current data on photosensitizer nanotransporters are presented. The potential sensors for reactive oxygen species in cells are discussed.

An acoustic method for the analysis of bacterial cells

Abstract: The application of a biological electroacoustic sensor based on a lateral electric-field-excited piezoelectric resonator for the study of bacterial cells that interact with specific bacteriophages, mini-antibodies, and polyclonal antibodies was successfully demonstrated. The determined lower limit of microbialcell detection was approximately of 103 to 104 cells/mL for the duration of the assay of 10 min. The possibility of bacterial-cell detection via interaction with specific agents in the presence of extraneous microbiota was shown. The method allowed us to determine the spectrum of lytic activity of bacteriophages and the sensitivity of microbial cells to bacteriophages. The results of the study showed that application of a sensor piezoelectric lateral-field resonator is a promising technique for the detection and identification of microbial cells and determination of their phage resistance in microbiology, medicine, and veterinary medicine. Furthermore, the results of the experiments made it possible to understand the mechanisms of the processes that occur in a suspension of bacterial cells in the presence of various biological agents. The method also may provide useful information regarding biophysical mechanisms of interactions that occur in microbial populations.

The effect of weak magnetic fields on the chemiluminescence of human blood

Abstract: Exposure of heparinized human venous blood that was diluted with a phosphate buffer to a combination of a static magnetic field (42 µT) and a weak (amplitude range 108–3440 nT) variable low-frequency (1, 4.4, and 16.5 Hz, ratio of amplitudes 6: 1: 1.6, respectively) magnetic field collinear to the static magnetic field enhanced blood chemiluminescence that was induced by the addition of luminol or lucigenin at physiological temperature. The free-radical scavenger edaravone (MCI-186) and apocynin, an inhibitor of NADPH oxidase, reduced the intensity of blood chemiluminescence and alleviated the effects of the magnetic fields.

Genetic analysis of revertants isolated from the rod-fragile fliF mutant of Salmonella

Abstract: FliF is the protein comprising the MS-ring of the bacterial flagellar basal body, which is the base for the assembly of flagellar axial structures. From a fliF mutant that easily releases the rod-hook-filament in viscous environments, more than 400 revertants that recovered their swarming ability in viscous conditions, were isolated. The second-site mutations were determined for approximately 70% of them. There were three regions where the mutations were localized: two in Region I, 112 in Region II, and 71 in Region III including the true reversion. In Region I, second-site mutations were found in FlgC and FlgF of the proximal rod, suggesting that they affect the interaction between the MS-ring and the rod. In Region II, there were 69 and 42 mutations in MotA and MotB, respectively, suggesting that the second-site mutations in MotA and MotB may decrease the rotational speed of the flagellar motor to reduce the probability of releasing the rod under this condition. One exception is a mutation in FlhC that caused a down regulation of the flagellar proteins production but it may directly affect transcription or translation of motA and motB. In Region III, there were 44, 24, and 3 mutations in FliG, FliM, and FliF, respectively. There were no second-site mutations identified in FliN although it is involved in torque generation as a component of the C-ring. Many of the mutations were involved in the motor rotation, and it is suggested that such reduced speeds result in stabilizing the filament attachment to the motor.

On the modeling of the motion of a transcription bubble under constant torque

Abstract: The motion of a transcription bubble under constant torque is investigated. The motion of the bubble was modeled by a modified sine-Gordon equation. Numerical solutions of this equation (kinks) were found. Trajectories of the kink motion in homogeneous and inhomogeneous sequences were calculated for different model values of the torque M τ and the initial velocity v 0. It was shown that a change in the torsion moment M τ has a significant impact on the character of the kink trajectory. At the same time, a change in the initial kink velocity v 0 in a rather wide range of values does not affect the character of the kink trajectory.

Characterization of lipodisc nanoparticles containing sensory rhodopsin II and its cognate transducer from Natronomonas pharaonis

Abstract: We describe the preparation and properties of lipodisc nanoparticles–lipid membrane fragments with a diameter of about 10 nm, stabilized by amphiphilic synthetic polymer molecules. We used the lipodisc nanoparticles made of Escherichia coli polar lipids and compared lipodisc nanoparticles that contained the photosensitive protein complex of the sensory rhodopsin with its cognate transducer from the halobacterium Natronomonas pharaonis with empty lipodisc nanoparticles that contained no protein. The lipodisc nanoparticles were characterized by dynamic light scattering, transmission electron microscopy and atomic force microscopy. We found that the diameter of lipodisc nanoparticles was not affected by incorporation of the protein complexes, which makes them a prospective platform for single-molecule studies of membrane proteins.

The role of electrostatic interactions in the formation of ferredoxin–ferredoxin NADP + reductase and ferredoxin–hydrogenase complexes

Abstract: A competitive Brownian model for the interaction of ferredoxin, ferredoxin NADP+ reductase and hydrogenase has been built. In the model, molecules of three types of proteins are placed into a cubic reaction volume, where they move under Brownian and electrostatic forces created by neighboring molecules and the solution. It has been shown that the rate of ferredoxin binding with ferredoxin NADP+ reductase does not change at the pH range from 5.0 to 9.0. Thus, it may be suggested that regulation of ferredoxin NADP+ reductase activity is mediated by other processes. On the other hand, the rate of ferredoxin binding with hydrogenase in the model depends greatly on pH: if the pH value increases from 6.0 to 8.0 the rate increases by factor of three. The increase of the pH value in the stroma under illumination results in an increase of the rate of its interaction with ferredoxin, but decreases the level of protons that are the substrate for the reaction catalyzed by the protein. Thus, the rate of hydrogen production in the chloroplast stroma is low at low pH due to the reception of a small number of electrons by hydrogenase. When the pH increases, the number of electrons that are received by the enzyme from ferredoxin also increases; thus, the rate of hydrogen production increases as well.

Application of the method of electro-acoustical analysis for the detection of bacteriophages in a liquid phase

Abstract: The possibility of the application of electro-acoustic analysis for the detection of bacteriophages was demonstrated for the first time based on the example of the interaction of the FA1-Sp59b bacteriophage with bacterial cells of the strain Azospirillum lipoferum Sp59b. Piezoelectric cross-field resonators with a 1-mL chamber for analyzed liquid were used as the biological sensor. It was revealed that the dependences of the real and imaginary parts of the electrical impedance of the resonator loaded with a suspension of viruses and microbial cells on the frequency was significantly different from those dependences of the resonator that contained a control cell suspension without the virus. It was shown that detection of the FA1-Sp59b bacteriophage using microbial cells was possible with both extraneous viral particles and extraneous microbial cells. The proposed method allows one to accurately determine the type of identified virus after a 5-minute interaction with indicating bacterial culture. As well, the minimum concentration of viruses is five virus particles per cell. These results as a whole demonstrate the possibility of detecting specific interactions of bacteriophages with microbial cells and provide a basis for the development of a biological sensor for the quantitative detection of viruses directly in the liquid phase.

Theoretical analyses on a flipping mechanism of UV-induced DNA damage.

Abstract: As for UV-induced DNA damage, which may induce skin cancer in animals and growth inhibition in plants, there are two types of photoproducts, namely cis-sin cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts. When they are to be repaired, base-flipping occurs, and they bind to enzymes. However, this process remains relatively unknown at a molecular level. We analyze conformation and interaction energy changes upon base-flipping using classical molecular dynamics (CMD) simulations and ab initio electronic structure calculations. CMD simulations starting with a CPD in the flipped-in and flipped-out states showed that both states were unchanged for 500 ns, indicating the flipped-in and flipped-out processes do not occur spontaneously (without any help of the enzyme) after photo-damage. To deeply understand the reasons, we investigated interaction energy changes among bases upon structure changes during the flipped-in and flipped-out processes using Parallel Cascade Selection-MD (PaCS-MD) simulations at 400 K, followed by a fragment molecular orbital (FMO) method. The total inter-fragment interaction energy (IFIE) between CPD and other bases at the flipped-in state is estimated to be -60.08 kcal/mol. In particular, four bases strongly interact with CPD with interaction energies being -10.96, -13.70, -21.52, and -14.46 kcal/mol each. On the other hand, the total IFIE at the obtained flipped-out state increased to -10.40 kcal/mol by partly losing hydrogen bonds and π-π stacking interactions, respectively. These results clearly indicate that the base-flipping process of DNA lesions occurs with the help of external forces like interactions with appropriate enzymes such as photolyases.