Co-operative intra-protein structural response due to protein–protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.     

Scrambled Geometric Net Integration Over General Product Spaces

Quasi-Monte Carlo (QMC) sampling has been developed for integration over \([0,1]^s\) where it has superior accuracy to Monte Carlo (MC) for integrands of bounded variation. Scrambled net quadrature allows replication-based error estimation for QMC with at least the same accuracy and for smooth enough integrands even better accuracy than plain QMC. Integration over triangles, spheres, disks and Cartesian products of such spaces is more difficult for QMC because the induced integrand on a unit cube may fail to have the desired regularity. In this paper, we present a construction of point sets for numerical integration over Cartesian products of s spaces of dimension d, with triangles (\(d=2\)) being of special interest. The point sets are transformations of randomized (t, m, s)-nets using recursive geometric partitions. The resulting integral estimates are unbiased, and their variance is o(1 / n) for any integrand in \(L^2\) of the product space. Under smoothness assumptions on the integrand, our randomized QMC algorithm has variance \(O(n^{-1 - 2/d} (\log n)^{s-1})\), for integration over s-fold Cartesian products of d-dimensional domains, compared to \(O(n^{-1})\) for ordinary MC.     

Response Factor Correction for Estimation of Ester Content in Biodiesel

EN 14103 is generally used for quantification of ester content in biodiesel free of heptadecanoate ester (C17:0) or methyl nonadecanoate (C19:0), which are employed as internal standards. It was observed that ester content obtained by EN 14103 method did not match with theoretical value of biodiesel, as the method did not take care of response factors of each component to compensate for changes in detector sensitivities. In this study, the whole range of fatty acid (C₆–C_24:1) methyl esters have been taken into consideration for the calculation of the ester content. Methyl nonadecanoate (C19:0) was used as an internal standard. The response factors of both the saturated and unsaturated methyl esters in the range C₆–C_24:1 were estimated and found in the range 0.97–1.16. The ester content was calculated after applying the response factors of each methyl ester. The results obtained by this method agreed well with the theoretical value as compared to estimated value using EN14103 method. The results obtained from this method also show good correlation (R ² = 0.98) with ¹H-NMR method. Further, this method does not depend on nature of biodiesel feed stock and is applicable to all methyl biodiesel samples obtained from different raw materials.     

Facile synthesis of substituted pyrrole-fused isocoumarins from ninhydrin

A simple and efficient procedure has been developed for the synthesis of substituted pyrrole-fused isocoumarins from easily available ninhydrin. The cyclic hemiaminal dihydroxy-indenopyrroles, the adducts of ninhydrin with enamines of acetylacetone, give pyrrole-fused isocoumarins upon heating in acidic medium. The process constitutes an interesting acid-catalyzed rearrangement to eight-membered lactams followed by intramolecular cyclization involving the amino and keto groups.     

Comparative determination of uranium in rock phosphates and columbite by ICP-OES,alpha &; gamma spectrometry

During this work selective separation of uranium from rock phosphate and columbite mineral was done before its quantitative estimation by using Inductively Coupled Plasma Optical Emission Spectrometery (ICP-OES). Uranium from the rock phosphate and columubite was extracted by sodium peroxide fusion followed by leaching in 2 M HNO₃. To avoid spectral interference in the estimation of uranium by ICP-OES, the selective separation of uranium from the leachate was carried out by using two different extractants, 30% Tributyl Phophates (TBP) in CCl₄ and a equi-volume mixture of Di(2-ethylhexyl) phosphoric acid (D2EHPA) & TBP in petrofin. Uranium was stripped from the organic phase by using 1 M ammonium carbonate solution. Determination of uranium by ICP-OES was done after dissolving the residue left after evaporation of ammonium carbonate solution in 4% HNO₃. The concentration of the uranium observed in the rock phosphates samples was 40–200 μg g⁻¹ whereas in columbite samples the concentration range was 100–600 μg g⁻¹. Uranium concentration evaluated by ICP-OES was complimented by gamma & alpha spectrometry. Concentration of uranium evaluated by gamma spectrometry in case of rock phosphate and coulmbite was in close agreement with the uranium content obtained by ICP-OES. Uranium determination by alpha spectrometry showed only minor deviation (1–2%) from the results obtained by ICP-OES in case of rock phosphates whereas in case of coulmbites results are off by 20–30%.     

In vitro evolution of ligands to the membrane protein caveolin

Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, although implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with >1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to caveolin by the T20 variants with K(d) values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.     

Conformational fluctuations of a protein-DNA complex and the structure and ordering of water around it

Protein-DNA binding is an important process responsible for the regulation of genetic activities in living organisms. The most crucial issue in this problem is how the protein recognizes the DNA and identifies its target base sequences. Water molecules present around the protein and DNA are also expected to play an important role in mediating the recognition process and controlling the structure of the complex. We have performed atomistic molecular dynamics simulations of an aqueous solution of the protein-DNA complex formed between the DNA binding domain of human TRF1 protein and a telomeric DNA. The conformational fluctuations of the protein and DNA and the microscopic structure and ordering of water around them in the complex have been explored. In agreement with experimental studies, the calculations reveal conformational immobilization of the terminal segments of the protein on complexation. Importantly, it is discovered that both structural adaptations of the protein and DNA, and the subsequent correlation between them to bind, contribute to the net entropy loss associated with the complex formation. Further, it is found that water molecules around the DNA are more structured with significantly higher density and ordering than that around the protein in the complex.     

Dynamic properties of water around a protein-DNA complex from molecular dynamics simulations

Formation of protein-DNA complex is an important step in regulation of genes in living organisms. One important issue in this problem is the role played by water in mediating the protein-DNA interactions. In this work, we have carried out atomistic molecular dynamics simulations to explore the heterogeneous dynamics of water molecules present in different regions around a complex formed between the DNA binding domain of human TRF1 protein and a telomeric DNA. It is demonstrated that such heterogeneous water motions around the complex are correlated with the relaxation time scales of hydrogen bonds formed by those water molecules with the protein and DNA. The calculations reveal the existence of a fraction of extraordinarily restricted water molecules forming a highly rigid thin layer in between the binding motifs of the protein and DNA. It is further proved that higher rigidity of water layers around the complex originates from more frequent reformations of broken water-water hydrogen bonds. Importantly, it is found that the formation of the complex affects the transverse and longitudinal degrees of freedom of surrounding water molecules in a nonuniform manner.     

2-(2-Pyridyl) benzimidazole based Co(II) complex as an efficient fluorescent probe for trace level determination of aspartic and glutamic acid in aqueous solution: a displacement approach

A weakly fluorescent cobalt(II) complex is synthesized using 2-(2-pyridyl)-benzimidazole (PBI) as a chelating fluorescent ligand and characterized by single crystal X-ray structure. This complex serves as an efficient fluorescent probe for trace level determination of aspartic acid (AspA) and glutamic acid (GluA) in aqueous solution. Rest of the naturally occurring amino acids did not interfere. Both aspartic acid and glutamic acid replaces PBI from the coordination sphere of Co(II)-PBI complex resulting appearance of strong fluorescence signal for the released free PBI. The signal response is very fast and the interaction of both the AspA and GluA with the Co(II) is strong enough as evident from their displacement equilibrium constant values, viz. 4357.8 M(-1) and 8333.33 M(-1) respectively.