Project description:In this work, the effect of two organic polyamines (spermine and spermidine) on the fluorescence intensity and activity of bovine intestinal alkaline phosphatase (BIALP) are investigated. The interaction of BIALP with spermine and spermidine was studied in a diethanolamine buffer with 0.5 mM magnesium chloride (pH 9.8) and at two temperatures by using the fluorescence quenching method. Furthermore, the activity of enzyme was studied using UV-Vis spectrophotometry in a diethanolamine buffer with 0.5 mM magnesium chloride, at 37 °C, in the absence and presence of different concentrations of each polyamine (0-5 mM). It was demonstrated that both polyamines quenched the intrinsic fluorescence of BIALP by the static quenching process. Based on these results, the values of the binding site for both polyamines were close to each other and decreased by increasing the temperature. The calculated thermodynamic parameters (ΔH° < 0 and ΔS° < 0) also showed that the acting forces in the formation of the complex between BIALP and polyamines were hydrogen bonds and van der Waals forces with an overall favorable Gibbs free energy change (∆G° < 0). In addition, kinetic studies revealed that these polyamines enhanced the enzyme activity of BIALP in a concentration-dependent manner. This result also indicated that spermine had more of an effect on BIALP activity in the same condition. Also, molecular docking as well as thermodynamic parameters showed that hydrogen bonds and van der Waals forces played an important role in the stabilization of BIALP-polyamine complexes.
Project description:Interactions between metals and activated sludge microorganisms substantially affect the speciation, immobilization, transport, and bioavailability of trace heavy metals in biological wastewater treatment plants. In this study, the interaction of Cu(II), a typical heavy metal, onto activated sludge microorganisms was studied in-depth using a multi-technique approach. The complexing structure of Cu(II) on microbial surface was revealed by X-ray absorption fine structure (XAFS) and electron paramagnetic resonance (EPR) analysis. EPR spectra indicated that Cu(II) was held in inner-sphere surface complexes of octahedral coordination with tetragonal distortion of axial elongation. XAFS analysis further suggested that the surface complexation between Cu(II) and microbial cells was the distorted inner-sphere coordinated octahedra containing four short equatorial bonds and two elongated axial bonds. To further validate the results obtained from the XAFS and EPR analysis, density functional theory calculations were carried out to explore the structural geometry of the Cu complexes. These results are useful to better understand the speciation, immobilization, transport, and bioavailability of metals in biological wastewater treatment plants.
Project description:A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis-Menten model and is inactivated by H2O2. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H2O2] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H2O2 decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H2O2 decomposition by catalase in 32 samples in triplicates if kept below 8 mM min-1 in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost.
Project description:An effort to combine theoretical analyses and protein engineering methods has been made to probe the folding mechanism of SH3 by using Energy Landscape Theory and a phi-value analysis. Particular emphasis was given to core residues and the effect of desolvation during the folding event by replacing the core valines with isosteric threonines. These mutations have the advantage of keeping the core structurally invariant while affecting core stability relative to the unfolded state. Although the valines that form the core appear spatially invariant, the folding kinetics of their threonine mutants varies, indicating their different extent of solvation in the transition-state ensemble. Theoretical studies predicted the distribution of folding kinetics of threonine mutants without previous knowledge of the measured rates. This initial success encourages further investigations of the molecular details behind these macroscopic phenomena and of the role of solvation in the folding mechanism.
Project description:Purpose: The study on the interaction between various compounds and macromolecules such as enzymes has been very important for monitoring the alteration of structural and functional properties of them. Resveratrol (3, 5, 4-trihydroxy-stilbene; RES) is a biologically active phytoallexin found in grapes and other food products. This article shows an interaction of native bovine liver catalase (BLC) with natural antioxidant product, trans resveratrol (tRES) using multispectroscopic methods. Methods: The interaction between BLC and tRES is performed using UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopy and molecular docking study. Results: In vitro kinetic studies indicated that tRES can decrease BLC activity through uncompetitive inhibition. The results of spectroscopic methods represented that the binding of tRES with BLC can change the micro-region around aromatic amino acids (tryptophan (Trp) and tyrosine (Tyr)) and quench intrinsic fluorescence of BLC by a static mechanism. According to fluorescence quenching data analysis, it was revealed that tRES has one binding site on BLC. The thermodynamic parameters were obtained, which demonstrated that tRES can bind to BLC by van der Waals forces and hydrogen bonds. Molecular docking results indicated that tRES binds to BLC away from heme group and near to the Tyr 324 and Phe 265. These results are in agreement with the experimental results. Conclusion: The inhibitory effect of tRES on BLC demonstrated that excessive consumption of the antioxidants could be resulted in hazardous effects.
Project description:Lawsone (2-hydroxy-1,4-naphtoquinone; LAW), as a naphthoquinone derivative, is the biologically active component of Henna leaves. In this study, the structural and functional effects of LAW on bovine liver catalase (BLC), has been studied utilizing ultraviolet-visible (UV-vis) absorption, fluorescence, and ATR-FTIR spectroscopic techniques, and molecular docking approach. In-vitro kinetic study showed that by adding gradual concentrations of LAW, catalase activity was significantly decreased through noncompetitive inhibition mechanism. UV-vis and ATR-FTIR spectroscopic results illustrated that additional concentration of LAW lead to significant change in secondary structure of the enzyme.The fluorescence spectroscopic results at different temperatures indicated that LAW quenches the intrinsic fluorescence of BLC by dynamic mechanismand there is just one binding site for LAW on BCL. Changing the micro-environment nearby two aromatic residues (tryptophan (Trp) and tyrosine (Tyr)) were resulted from synchronous fluorescence. The thermodynamic parameters were implied that the hydrophobic bindings have a significant impress in the organization of the LAW-catalase complex. Molecular docking data in agreement with experimental results, confirmed that hydrophobic interactions are dominant. Inhibition of enzyme activity by LAW, showed that along withits helpful effects as ananti-oxidant compounds, the side effects of LAW should not be overlooked.
Project description:Insulin-like growth factor binding protein 7 (IGFBP7) can bind to insulin with high affinity which inhibits the early steps of insulin action. Lack of recognition mechanism impairs our understanding of insulin regulation before it binds to insulin receptor. Here we combine computational simulations with experimental methods to investigate the interaction between IGFBP7 and insulin. Molecular dynamics simulations indicated that His200 and Arg198 in IGFBP7 were key residues. Verified by experimental data, the interaction remained strong in single mutation systems R198E and H200F but became weak in double mutation system R198E-H200F relative to that in wild-type IGFBP7. The results and methods in present study could be adopted in future research of discovery of drugs by disrupting protein-protein interactions in insulin signaling. Nevertheless, the accuracy, reproducibility, and costs of free-energy calculation are still problems that need to be addressed before computational methods can become standard binding prediction tools in discovery pipelines.
Project description:The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.
Project description:BackgroundProcessing and analysis of DNA sequences obtained from next-generation sequencing (NGS) face some difficulties in terms of the correct prediction of DNA sequencing outcomes without the implementation of bioinformatics approaches. However, algorithms based on NGS perform inefficiently due to the generation of long DNA fragments, the difficulty of assembling them and the complexity of the used genomes. On the other hand, the Sanger DNA sequencing method is still considered to be the most reliable; it is a reliable choice for virtual modeling to build all possible consensus sequences from smaller DNA fragments.ResultsIn silico and in vitro experiments were conducted: (1) to implement and test our novel sequencing algorithm, using the standard cloning vectors of different length and (2) to validate experimentally virtual shotgun sequencing using the PCR technique with the number of cycles from 1 to 9 for each reaction.ConclusionsWe applied a novel algorithm based on Sanger methodology to correctly predict and emphasize the performance of DNA sequencing techniques as well as in de novo DNA sequencing and its further application in synthetic biology. We demonstrate the statistical significance of our results.
Project description:Synthesis and characterization of substituted 2,6-dibenzylidene cyclohexanone-based bischalcone derivatives and their optimized geometries were investigated by density functional theory. The synthesized compounds were identified through ultraviolet-visible, Fourier transform infrared, and 1H nuclear magnetic resonance spectroscopies and elemental analysis. Significant acidochromic behavior was observed for 2,6-bis(4-dimethylamino-benzylidene)-cyclohexanone 1e. This result is owing to the preferential protonation of the chromophoric N,N-dimethylamino group, that is, quaternary salt formation and deactivation of the resonance system. The result was consistent with computational studies where the protonation was favored by 211 kcal/mol in the gas phase. The compounds also showed solvatochromic behavior. The geometries of the synthesized compounds were optimized with B3LYP/6-311G+(d,p) and APFD/6-311+G(d,p) basis sets. The single point energy indicated that APFD/6-311+G(d,p) basis set gave the lowest energy of 445-655 kcal/mol for the studied bischalcone derivatives. Quantum chemical parameters were also calculated.