Loop Interactions during Catalysis by Dihydrofolate Reductase fromMoritella profunda

Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the wellstudied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR

A kinetic model for alkaline leaching of uranium from dolomitic limestone ore

The choice of type of leaching to be employed in the leaching process for extraction of uranium from its ore is dictated by the chemical nature of gangue minerals rather than that of uranium minerals present in the ore. The kinetics of extraction of uranium values into aqueous phase is dependent on type of leachant chosen, uranium minerals present and their mode of occurrence. Present study is aimed to get an insight into the kinetics of alkaline leaching of uranium from an Indian uranium ore containing high carbonates, about 80%, as gangue minerals. Heterogeneous kinetic model, shrinking core with mixed surface reaction and diffusion dissolution kinetics was found to be best fitting to predict the rate of leaching. The ranges of influence of factors studied were partial pressure of oxygen from 4.5 kg/cm2 to 6.5 kg/cm2, stirring speed between 573 rpm to 900 rpm, reaction temperature in the range 125°C to 165°C and particle size from 25.8 µ to 34.76 µ. The orders of reaction with respect to partial pressure of oxygen, stirring speed, particle size and Arrhenius activation energy of uranium leaching were determined to be 2.5, 0.21, –5.7 and 4.5 kcal/mole respectively

NMR Metabolomics Protocols for Drug Discovery

Drug discovery is an extremely difficult and challenging endeavor with a very high failure rate. The task of identifying a drug that is safe, selective and effective is a daunting proposition because disease biology is complex and highly variable across patients. Metabolomics enables the discovery of disease biomarkers, which provides insights into the molecular and metabolic basis of disease and may be used to assess treatment prognosis and outcome. In this regard, metabolomics has evolved to become an important component of the drug discovery process to resolve efficacy and toxicity issues, and as a tool for precision medicine. A detailed description of an experimental protocol is presented that outlines the application of NMR metabolomics to the drug discovery pipeline. This includes: (1) target identification by understanding the metabolic dysregulation in diseases, (2) predicting the mechanism of action of newly discovered or existing drug therapies, (3) and using metabolomics to screen a chemical lead to assess biological activity. Unlike other OMICS approaches, the metabolome is “fragile”, and may be negatively impacted by improper sample collection, storage and extraction procedures. Similarly, biologically-irrelevant conclusions may result from incorrect data collection, pre-processing or processing procedures, or the erroneous use of univariate and multivariate statistical methods. These critical concerns are also addressed in the protocol

Microstructure and mechanical properties of cryorolled aluminum alloy AA2219 in different thermomechanical processing conditions

In the present study, aluminum alloy AA2219-T87 bars were cryorolled to various amounts of deformation in two pre-deformation conditions: (1) without solution treatment i.e., as-received T87 (WST-CR) and (2) with solution treatment (ST + CR). The solution treated and cryorolled bars were further annealed leading to a third condition: (3) solution treated, cryorolled, and annealed (CR + Annealed). Room-temperature mechanical properties have been evaluated for all three cryorolled conditions. Significant improvement in the 0.2 pct YS and UTS values was obtained for bars cryorolled to cross-sectional area reduction of more than 50 pct in the solution-treated condition (ST + CR), whereas for bars cryorolled in the without solution-treated condition (WST-CR), only an improvement in the 0.2 pct YS was observed. Cryorolling did not enhance the precipitation kinetics nor did it increase the response of the alloy to aging. The mechanical properties were correlated to the microstructures obtained by optical and transmission electron microscopy. Microstructural evolution in the ST + CR condition indicated gradual progression of the principal restoration mechanism from dynamic recovery (DRV) to dynamic recrystallization with an increasing amount of plastic deformation. Transmission electron microscopy of WST-CR and ST + CR specimens showed an increase in dislocation density as a function of the amount of deformation indicating suppression of DRV at cryogenic temperatures. Cryorolling in the solution-treated condition to cross-sectional area reduction of more than 50 pct (ST + 70 pct CR) was found to impart an optimum combination of strength and percent elongation in the present study.by Aditya Sarkar, K. Saravanan, Niraj Nayan, S. V. S. Narayana Murty, P. Ramesh Narayanan, P. V. Venkitakrishnan and Jyoti Mukhopadhya

Development of processing maps and constitutive relationship for thermomechanical processing of Aluminum Alloy AA2219

Isothermal uniaxial compression tests were conducted on aluminum alloy AA2219 to study the evolution of microstructure over a wide range of temperatures (300-500 °C) and strain rates (0.001-100 s−1) with a view to study the flow behavior and concurrent microstructural evolution. True stress-true strain curves showed only a gradual flow softening at all temperatures except at 300 °C where strain hardening was followed by severe flow softening. Processing map delineating the stable ‘safe’ and unstable ‘unsafe’ regions during hot working is developed and validated by comparing the microstructures observed in the deformed compression specimens. Optimum processing parameters (temperature 450 °C and strain rate 0.001 s−1) for hot deformation of AA2219 were proposed based on contour maps of efficiency of power dissipation and strain rate sensitivity parameter. The activation energy value (Qavg) of AA2219 for hot working was computed to be 169 kJ/mol. Finally, a constitutive equation for hot working of AA2219 was established as: ε˙=4.99×109⋅exp(0.06149σ)⋅exp(−168.958/RT)ε˙=4.99×109⋅exp⁡(0.06149σ)⋅exp⁡(−168.958/RT).by S. V. S. Narayana Murty, Aditya Sarkar, P. Ramesh Narayanan, P. V. Venkitakrishnan and Jyoti Mukhopadhya

The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR