289 NUMBERFIGURE 7. Representative isothermal titration calorimetry for the binding of 1-stearoyl-rac-glycerol to
289 NUMBERFIGURE 7. Representative isothermal titration calorimetry for the binding of 1-stearoyl-rac-glycerol to Rv0678. a, every peak corresponds for the injection of 10 l of 200 M dimeric Rv0678 in buffer containing 10 mM sodium phosphate (pH 7.two), 100 mM NaCl, and 0.001 n-dodecyl- -maltoside into the reaction containing 10 M 1-stearoyl-rac-glycerol inside the exact same buffer. b, cumulative heat of reaction is displayed as a function of your injection quantity. The Phospholipase A Synonyms strong line will be the least square match towards the experimental data, providing a Ka of four.9 0.4 105 M 1.The propanetriol in the bound 2-stearoylglycerol is totally buried within the dimer interface, leaving the tail portion of its elongated octadecanoate hydrophobic carbon chain oriented in the entry point of this binding web page. This orientation facilitates the contribution of Arg-32 and Glu-106 to form two hydrogen bonds with all the glycerol headgroup of the fatty acid. The backbone oxygen of Phe-79 also participates to make the third hydrogen bond with this glycerol headgroup. In addition, the carbonyl oxygen in the octadecanoate group contributes to produce one more hydrogen bond with Arg-109, securing the binding. Interestingly, Rv0678 further anchors the bound fatty acid molecule via hydrophobic interactions with residues Phe79, Phe-79 , and Phe-81 . Thus, the binding of 2-stearoylglycerol in Rv0678 is substantial; within four.five of the bound fatty acid glycerol ester, 20 amino acids speak to this molecule (Table 4). It must be noted that residues Phe-79, Phe-79 , and Phe81 belong to helices four and four . In the OhrR-DNA structure (36), the corresponding 4 and four helices were buried inside the two consecutive major grooves, straight contacting the promoter DNA. As a result, we suspect that helices 4 and 4 have dualJOURNAL OF BIOLOGICAL CHEMISTRYStructure from the Transcriptional Regulator RvFIGURE eight. Rv0678 binds to promoter regions of mmpS2-mmpL2, mmpS4-mmpL4, mmpS5, and rv0991c. a, schematic depicting the DNA probes applied in EMSAs to examine the promoter and intragenic regions with the mmpS2-mmpL2, mmpL3, mmpS4-mmpL4, mmpS5-mmpL5, and rv0991-2c genes. b, EMSAs have been performed working with 12 nM DIG-labeled probe as well as the indicated micromolar concentrations of protein. An arrow denotes the shifted probes. c, to demonstrate specificity, EMSAs had been performed within the mGluR1 custom synthesis presence of non-labeled (“cold”) probe. Reactions were performed with six nM DIG-labeled probe, the indicated micromolar concentrations of protein, and 0.6 M cold probe. *, accumulation of cost-free DIG-labeled probe. d, EMSAs have been performed working with 12 M DIG-labeled probe and six M Rv0678 in the presence or absence of 1 M 1-stearoyl-rac-glycerol, as indicated above the blot. e, the sequence on the probes bound by Rv0678 in b and c were compared applying the motif-based sequence evaluation tool MEME, yielding a putative Rv0678 binding motif.responsibilities within the Rv0678 regulator. They form the DNAbinding web page for operator DNA too because the substrate-binding site for inducing ligands. Inside the second Rv0678 dimer with the asymmetric unit, it’s also discovered that a 2-stearoylglycerol molecule is bound inside the corresponding substrate-binding website. Residues contributed to type this binding web-site are almost identical but using a slightly distinct subset of amino acids in comparison with these of the initially Rv0678 dimer described above (Table four). Virtual Ligand Library Screening–Virtual ligand screening was then performed to elucidate the nature of protein-ligand interactions in.