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Mutational Studies (mutational + studies)
Selected AbstractsMutational Studies Confirm the Catalytic Triad in the Human Selenoenzyme Thioredoxin Reductase Predicted by Molecular ModelingCHEMBIOCHEM, Issue 11 2006Stephan Gromer Dr. Three's company. Site-directed mutagenesis of Glu477 of the human thioredoxin reductase (see figure) to glutamine, alanine, or lysine led to a significant drop in enzymatic activity. This study reinforces previous theoretical calculations which suggested that a swapping catalytic triad exists in the active site of this enzyme. [source] Involvement of nuclear factor-kappa B in bcl-xL-induced interleukin 8 expression in glioblastomaJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Chiara Gabellini Abstract We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkB, and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkB, protein, paralleled by an increase in the phosphorylation of the same IkB, and IKK,/,. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkB, in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells. [source] Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognitionGENES TO CELLS, Issue 5 2000Masayuki Oda Thermodynamic and kinetic analyses of biomolecular interactions reveal details of the energetic and dynamic features of molecular recognition processes, and complement structural analyses of the free and complexed conformations. The recent improvements in both isothermal titration calorimetry and surface plasmon resonance sensoring provide powerful tools for analysing biomolecular interactions in thermodynamic and kinetic approaches. The thermodynamic and kinetic parameters obtained for binding between protein and DNA indicate the mechanism of specific DNA recognition, in the high-resolution structures of the protein,DNA complexes. The effects of temperature and ionic strength reflect the conformational changes of the protein and DNA molecules upon complex formation, including important contributions of water and solutes. When combined with mutational studies, the interactions can be reduced to several energetic contributions from individual contacts. These studies should be useful to determine general features of protein functions in genetic regulation. [source] Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three-metal ion assisted catalysis and lithium inhibitionPROTEIN SCIENCE, Issue 2 2010Zheng Li Abstract The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg2+ for activity and binds three to four ions tightly in the absence of ligands: KD = 0.8 ,M for one ion with a KD of 38 ,M for the other Mg2+ ions. However, the enzyme requires 5,10 mM Mg2+ for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic KD for Mg2+; D26A, E39A, and E41A showed no significant change in the Mg2+ requirement for optimal activity. D38A also showed an increased Km, but little effect on kcat. This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg2+ in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC50,250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li+ with an IC50 of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily. [source] The combination of molecular dynamics with crystallography for elucidating protein,ligand interactions: a case study involving peanut lectin complexes with T-antigen and lactoseACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001J. V. Pratap Peanut lectin binds T-antigen [Gal,(1,3)GalNAc] with an order of magnitude higher affinity than it binds the disaccharide lactose. The crystal structures of the two complexes indicate that the higher affinity for T-antigen is generated by two water bridges involving the acetamido group. Fresh calorimetric measurements on the two complexes have been carried out in the temperature range 280,313,K. Four sets of nanosecond molecular-dynamics (MD) simulations, two at 293,K and the other two at 313,K, were performed on each of the two complexes. At each temperature, two somewhat different protocols were used to hydrate the complex in the two runs. Two MD runs under slightly different conditions for each complex served to assess the reliability of the approach for exploring protein,ligand interactions. Enthalpies based on static calculations and on MD simulations favour complexation involving T-antigen. The simulations also brought to light ensembles of direct and water-mediated protein,sugar interactions in both the cases. These ensembles provide a qualitative explanation for the temperature dependence of the thermodynamic parameters of peanut lectin,T-antigen interaction and for the results of one of the two mutational studies on the lectin. They also support the earlier conclusion that the increased affinity of peanut lectin for T-antigen compared with that for lactose is primarily caused by additional water bridges involving the acetamido group. The calculations provide a rationale for the observed sugar-binding affinity of one of the two available mutants. Detailed examination of the calculations point to the need for exercising caution in interpreting results of MD simulations: while long simulations are not possible owing to computational reasons, it is desirable to carry out several short simulations with somewhat different initial conditions. [source] Structures of the B1 domain of protein L from Peptostreptococcus magnus with a tyrosine to tryptophan substitutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2001Jason W. O'Neill The three-dimensional structure of a tryptophan-containing variant of the IgG-binding B1 domain of protein L has been solved in two crystal forms to 1.7 and 1.8,Å resolution. In one of the crystal forms, the entire N-terminal histidine-tag region was immobilized through the coordination of zinc ions and its structural conformation along with the zinc coordination scheme were determined. However, the ordering of the histidine tag by zinc does not affect the overall structure of the rest of the protein. Structural comparisons of the tryptophan-containing variant with an NMR-derived wild-type structure, which contains a tyrosine at position 47, reveals a common fold, although the overall backbone root-mean-square difference is 1.5,Å. The Y47W substitution only caused local rearrangement of several side chains, the most prominent of which is the rotation of the Tyr34 side chain, resulting in a 6,Å displacement of its hydroxyl group. A small methyl-sized cavity bounded by ,-strands 1, 2 and 4 and the ,-helix was found in the structures of the Y47W-substituted protein L B1 domain. This cavity may be created as the result of subsequent side-chain rearrangements caused by the Y47W substitution. These high-resolution structures of the tryptophan-containing variant provide a reference frame for the analysis of thermodynamic and kinetic data derived from a series of mutational studies of the protein L B1 domain. [source] | |||||||||||||