Charge-Pairing Interactions Control The Conformational Setpoint and Motions of The FMN Domain in Neuronal Nitric Oxide Synthase

The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu(762), Glu(816) and Glu(819)) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [K-eq(A)], of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5-4-fold) and rates of interflavin electron transfer (1.5-2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations

Distinct Conformational Behaviors of Four Mammalian Dual-Flavin Reductases (Cytochrome P450 Reductase, Methionine Synthase Reductase, Neuronal Nitric Oxide Synthase, Endothelial Nitric Oxide Synthase) Determine Their Unique Catalytic Profiles

Multidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase, methionine synthase reductase and endothelial and neuronal nitric oxide synthase. We used stopped-flow spectroscopy, single turnover methods and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. The results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12% to 72% open state) and also vary 100-fold with respect to their rates of conformational switching between unreactive closed and reactive open states (cytochrome P450 reductase \u3e neuronal nitric oxide synthase \u3e methionine synthase reductase \u3e endothelial nitric oxide synthase). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (cytochrome c reductase activity) based on its unique conformational setpoint and switching rates. The present study is the first to quantify these conformational parameters among the diflavin enzymes and suggests how the parameters might be manipulated to speed or slow biological electron flux

Cost-effective management of ufra disease of rice and identification of resistant landraces

A series of trials were undertaken to evaluate 3 nematicides, marshal 6G, diafuran 5G and pilarfuran 5G @ 1.0 kg ai/ha along with standard cheek, furadan 5G and to explore the resistant genotypes against ufra disease caused by Ditylenchus angustus in the rain-fed and irrigated ecosystems during 2001 to 2004. All the tested nematicides were effective to control the ufra disease of rice and increased yield compared to control (diseased). In respect of all seasons, marshal 6G, diafuran 5G, pilarfuran 5G increased yield by 3.35 to 5.10, 3.23 to 5.00 and 3.26 to 4.90 t/ha, respectively over the control (diseased). Yield loss due to ufra disease was 87.85% in the rain-fed rice, while it was 90.82% in the irrigated rice in artificial inoculation condition. In simple profitability analysis, marshal 6G, diafuran 5G and pilarfuran 5G showed 16.20, 15.76 and 15.58 times profitable in the rain-fed rice and 20.40, 20.11, 19.68 and 20.58 times profitable respectively, over the control (diseased) in the irrigated rice. So, the application of 3 nematicides, marshal 6G, diafuran 5G and pilarfuran 5G @ 1k ai/ha were effective in controlling ufra disease and could be used as alternative to furadan 5G. Of 40 landraces of rice tested, 5 (Daudin Da-21, Lambo Sail, Madhu Sail, Bhawalia Aman and Lal Chamara) showed highly resistant against ufra disease

Human visual perception inspired background subtraction

Background subtraction is an essential processing task for moving foreground detection. Existing approaches are reliable only when scenario-specific configuration is possible; otherwise, they exhibit highly unpredictable performance across a wide range of dynamic scenarios due to extensive dependence on statistical observations and context-specific constraints while lacking means for exploiting perceptual characteristics of the operating environment. This thesis investigates a theoretical framework for developing a novel human visual perception inspired background subtraction technique for unconstraint video analytics. The technique, named perceptual mixture-of-Gaussians (PMOG), emphasises on several perceptual characteristics of observed statistics for better exploitation of the operating environment to exhibit robustness in dynamic unconstrained scenarios. For instance, how human visual system perceives noticeable intensity deviation from the background; what is the perceptual tolerance level in distinguishing distorted intensity measures; and how realistic predictions can be made regarding an observation are the key questions investigated in the thesis. Addressing these questions has enabled PMOG to ensure high performance stability with superior detection quality across dynamic scenarios as well as optimal computational resource utilisation throughout the system lifetime. PMOG is then modified to improve responsiveness in unconstrained scenarios by incorporating a low-cost estimator of suspected foreground activities. The detection quality of PMOG is further enhanced by integrating two independent hypotheses originating from the same underlying model to maximise complementary aspects and minimise computational overhead. Comprehensive experimental evaluation is performed to establish superiority of PMOG against the state-of-the-art. Finally, the efficacy of PMOG is validated in the application domain of event detection

Oral 1832-2 – Charge Pairing and Phosphorylation Regulate The Conformational Equilibrium and Switching Rates in Neuronal and Endothelial NO Synthase Flavoprotein Domains

Electron flux through nitric oxide synthase reductase (NOSr) is thought to depend on conformational switching motions of their FMN domains, which enables the enzymes to cycle between closed unreactive and open reactive conformational states. However, the conformational equilibrium setpoints (Keq), rates of conformational switching, and interflavin electron transfer rates are mostly unknown, and how these parameters may combine to determine catalytic activities in NOSs is not well understood. To address these, we determined and compared the conformational equilibrium setpoints and rates of conformational switching between reactive open and unreactive closed states, in wild-type nNOSr and four FMN surface mutants (E762R, E762N, E816R, E819R) of nNOSr, and in wild-type eNOSr and the phospho-mimetic S1179D eNOSr mutant. We used stopped flow spectroscopy, single turnover methods, and a kinetic model that relates conformational setpoint and rates of conformational switching to the electron flux through each enzyme to cytochrome c. We found that charge neutralization or reversal at each of these residues alters the setpoint (Keq) of the NOSr conformational equilibrium to favor the open reactive (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the nNOSr mutants suggest that they have higher conformational transition rates (1.5–4-fold) relative to wild-type nNOSr. Wild-type eNOSr mostly exists in closed conformational state (88% closed, 12% open, Keq = 0.125) with a very slow electron flux. In comparison, the S1179D mutation alters the eNOSr setpoint to Keq = 1.5 (40% closed, 60% open), indicating that the open reactive conformation is favored in S1179D eNOSr. Our computer simulation data suggest that S1179D eNOSr also has a faster conformational transition, and a 20-fold faster opening rate relative to wild-type eNOSr. Thus, mutating Ser1179 to Asp alters both the setpoint and transition rates of equilibrium, and these can fully explain the increased electron flux seen in S1179D eNOSr mutant. Together, our studies provide the first measures of conformational equilibrium settings and conformational switching rates in nNOSr and eNOSr proteins, reveal that remarkable differences exist between the two proteins, and show how charge pairing interactions at the domain interface, or phosphorylation at Ser1179, alter NOS activity by modifying these conformational parameters

P101 – Endothelial Nitric Oxide Synthase (eNOS) on Lipid Nanodiscs: Toward A Soluble Assembly Reflecting Native-Like Function of eNOS

Cardiovascular disease (CVD) is the leading cause of death worldwide. Approximately 30% of all global deaths in 2008 were due to CVD. Endothelial cells cover the blood vessels lumen and provide a barrier against vascular disease. Nitric oxide is a unique bio-regulator with important signaling roles in cardiovascular as well as other physiologic systems. Nitric oxide synthases (NOSs) are a family of enzymes that generate nitric oxide from arginine and oxygen. Endothelial nitric oxide synthase (eNOS) is one member of this family, and is the dominant isoform in the inner walls of blood vessels. It regulates numerous essential cardiovascular functions including vasodilation (blood pressure), inhibition of platelet aggregation and adhesion to the vascular wall, which prevents atherosclerosis and unwanted blood clots. To determine the influence of the phospholipid bilayer on the structure and activity of eNOS in a defined system, we have incorporated the recombinant oxygenase subunit of the enzyme into miniature lipid membranes called nanodiscs which are 12.9 nm in diameter. These nanodiscs based on membrane scaffold proteins provide a unique system that mimics the enzyme\u27s native microenvironment, yet the prepared enzyme/nanodisc assemblies can be conveniently studied in solution like any soluble enzyme preparation. Homogenous eNOS/nanodisc samples are purified using size exclusion chromatography. The average size of nanodisc diameter was confirmed by particle analysis based on dynamic light scattering. Griess assay is used to measure activity of free and nanodisc-bound enzymes. As compared to the free enzyme, the specific activity of nanodisc-bound eNOS oxygenase appears to be much lower. These data suggest that the membrane environment affects the catalytic properties of eNOS oxygenase

Effect of sodium nitroprusside on H⁺ -ATPase activity and ATP concentration in <i>Candida albicans</i>

873-879ATP hydrolysis by plasma membrane H+-ATPase from Candida albicans has been investigated in presence of nitric oxide and various nutrients (sugars and amino acids). Sodium nitroprusside (SNP) was used as nitric oxide donor. It was found that ATP concentration decreased in SNP treated cells which was more in presence of sugars like glucose, xylose and 2-deoxy-D-glucose and amino acids as compared to their respective controls. The activity of H+-ATPase from plasma membrane decreased by 70 % in SNP treated cells. Both in vivo and in vitro treatments of SNP showed almost similar effects of decrease in ATPase activity. Effect of SNP was more pronounced in presence of nutrients. Interestingly, it was observed that vanadate did not show any independent effect in presence of nitric oxide. Several workers have reported similar type of results with other P-type A TPases. For the first time, it was observed in the present study that in presence of nitric oxide, H+-ATPase activity decreased like other P-type ATPases. Our study indicated that NO had a significant effect on ATP synthesis and activity of H+- ATPase. In the presence of NO, the ATP concentration was decreased indicating it affected mitochondrial electron transport chain. It may be concluded that NO, not only affects (inhibit) mitochondrial electron transport chain but also interferes with H+- ATPase of plasma membrane by changing its conformation resulting in decreased activity