The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Richard D. Kolodner - One of the best experts on this subject based on the ideXlab platform.
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engineered disulfide forming amino acid substitutions interfere with a Conformational Change in the mismatch recognition complex msh2 msh6 required for mismatch repair
Journal of Biological Chemistry, 2012Co-Authors: Victoria V Hargreaves, Christopher D Putnam, Richard D. KolodnerAbstract:Abstract ATP binding causes mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces Conformational Changes in Msh2-Msh6; however, the nature of these Conformational Changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a Conformational Change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue R1124. An engineered disulfide bond within this region prevented the ATP-driven Conformational Change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven Conformational Change plays a role in mismatch repair.
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Engineered disulfide-forming amino acid substitutions interfere with a Conformational Change in the mismatch recognition complex Msh2-Msh6 required for mismatch repair.
The Journal of biological chemistry, 2012Co-Authors: Victoria V Hargreaves, Christopher D Putnam, Richard D. KolodnerAbstract:ATP binding causes the mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces Conformational Changes in Msh2-Msh6; however, the nature of these Conformational Changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a Conformational Change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue Arg(1124). An engineered disulfide bond within this region prevented the ATP-driven Conformational Change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven Conformational Change plays a role in mismatch repair.
Gary Rudnick - One of the best experts on this subject based on the ideXlab platform.
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Substrate-Induced Conformational Change in LeuT
Biophysical Journal, 2017Co-Authors: Yuan-wei Zhang, Lucy R. Forrest, Gary RudnickAbstract:LeuT is a prokaryotic amino acid transporter that has been used extensively as a model for neurotransmitter transport. We recently demonstrated that the Conformational Change induced by Na+ ions requires the Na2 site observed in LeuT crystal structures. We observed this Conformational Change using LeuT in E. coli membranes, in the absence of detergent, by a decrease in reactivity of a single cysteine (Y265C) in the cytoplasmic permeation pathway. We now show that this effect of Na+ is observed whether K+ or NMDG+ is used as a control ion. In the presence of Na+, addition of alanine, a substrate, induces the reverse Conformational Change, opening the cytoplasmic pathway and increasing Cys-265 reactivity. Three mutations in the substrate binding site each altered the affinity of LeuT for leucine and alanine, but did not interfere with the Conformational Change induced by Na+. One of the mutations also blocked the substrate-dependent Conformational Change. The results suggest a mechanism by which substrate interactions with the central binding site of LeuT reverse the ability of Na+ to stabilize outward-facing conformations of this model transporter.
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two na sites control Conformational Change in a neurotransmitter transporter homolog
Journal of Biological Chemistry, 2016Co-Authors: Sotiria Tavoulari, Lucy R. Forrest, Eleonora Margheritis, Anu Nagarajan, David C. Dewitt, Yuan-wei Zhang, Edwin Rosado, Silvia Ravera, Elizabeth Rhoades, Gary RudnickAbstract:In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open Conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this Conformational Change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent Conformational Change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences Conformational Change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.
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Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog.
The Journal of biological chemistry, 2015Co-Authors: Sotiria Tavoulari, Lucy R. Forrest, Eleonora Margheritis, Anu Nagarajan, David C. Dewitt, Yuan-wei Zhang, Edwin Rosado, Silvia Ravera, Elizabeth Rhoades, Gary RudnickAbstract:In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open Conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this Conformational Change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent Conformational Change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences Conformational Change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.
Victoria V Hargreaves - One of the best experts on this subject based on the ideXlab platform.
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engineered disulfide forming amino acid substitutions interfere with a Conformational Change in the mismatch recognition complex msh2 msh6 required for mismatch repair
Journal of Biological Chemistry, 2012Co-Authors: Victoria V Hargreaves, Christopher D Putnam, Richard D. KolodnerAbstract:Abstract ATP binding causes mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces Conformational Changes in Msh2-Msh6; however, the nature of these Conformational Changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a Conformational Change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue R1124. An engineered disulfide bond within this region prevented the ATP-driven Conformational Change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven Conformational Change plays a role in mismatch repair.
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Engineered disulfide-forming amino acid substitutions interfere with a Conformational Change in the mismatch recognition complex Msh2-Msh6 required for mismatch repair.
The Journal of biological chemistry, 2012Co-Authors: Victoria V Hargreaves, Christopher D Putnam, Richard D. KolodnerAbstract:ATP binding causes the mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces Conformational Changes in Msh2-Msh6; however, the nature of these Conformational Changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a Conformational Change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue Arg(1124). An engineered disulfide bond within this region prevented the ATP-driven Conformational Change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven Conformational Change plays a role in mismatch repair.
Mitsunori Ikeguchi - One of the best experts on this subject based on the ideXlab platform.
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mechanism of the αβ Conformational Change in f1 atpase after atp hydrolysis free energy simulations
Biophysical Journal, 2015Co-Authors: Yuko Ito, Mitsunori IkeguchiAbstract:One of the motive forces for F1-ATPase rotation is the Conformational Change of the catalytically active β subunit due to closing and opening motions caused by ATP binding and hydrolysis, respectively. The closing motion is accomplished in two steps: the hydrogen-bond network around ATP Changes and then the entire structure Changes via B-helix sliding, as shown in our previous study. Here, we investigated the opening motion induced by ATP hydrolysis using all-atom free-energy simulations, combining the nudged elastic band method and umbrella sampling molecular-dynamics simulations. Because hydrolysis requires residues in the α subunit, the simulations were performed with the αβ dimer. The results indicate that the large-scale opening motion is also achieved by the B-helix sliding (in the reverse direction). However, the sliding mechanism is different from that of ATP binding because sliding is triggered by separation of the hydrolysis products ADP and Pi. We also addressed several important issues: 1), the timing of the product Pi release; 2), the unresolved half-closed β structure; and 3), the ADP release mechanism. These issues are fundamental for motor function; thus, the rotational mechanism of the entire F1-ATPase is also elucidated through this αβ study. During the Conformational Change, conserved residues among the ATPase proteins play important roles, suggesting that the obtained mechanism may be shared with other ATPase proteins. When combined with our previous studies, these results provide a comprehensive view of the β-subunit Conformational Change that drives the ATPase.
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mechanism of the Conformational Change of the f1 atpase β subunit revealed by free energy simulations
Journal of the American Chemical Society, 2011Co-Authors: Yuko Ito, Tomotaka Oroguchi, Mitsunori IkeguchiAbstract:F1-ATPase is an ATP-driven rotary motor enzyme. The β subunit Changes its conformation from an open to a closed form upon ATP binding. The motion in the β subunit is regarded as a major driving force for rotation of the central stalk. In this Article, we explore the Conformational Change of the β subunit using all-atom free energy simulations with explicit solvent and propose a detailed mechanism for the Conformational Change. The β subunit Conformational Change is accomplished roughly in two characteristic steps: changing of the hydrogen-bond network around ATP and the dynamic movement of the C-terminal domain via sliding of the B-helix. The details of the former step agree well with experimental data. In the latter step, sliding of the B-helix enhances the hydrophobic stabilization due to the exclusion of water molecules from the interface and improved packing in the hydrophobic core. This step contributes to a decrease in free energy, leading to the generation of torque in the F1-ATPase upon ATP binding.
Christopher D Putnam - One of the best experts on this subject based on the ideXlab platform.
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engineered disulfide forming amino acid substitutions interfere with a Conformational Change in the mismatch recognition complex msh2 msh6 required for mismatch repair
Journal of Biological Chemistry, 2012Co-Authors: Victoria V Hargreaves, Christopher D Putnam, Richard D. KolodnerAbstract:Abstract ATP binding causes mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces Conformational Changes in Msh2-Msh6; however, the nature of these Conformational Changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a Conformational Change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue R1124. An engineered disulfide bond within this region prevented the ATP-driven Conformational Change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven Conformational Change plays a role in mismatch repair.
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Engineered disulfide-forming amino acid substitutions interfere with a Conformational Change in the mismatch recognition complex Msh2-Msh6 required for mismatch repair.
The Journal of biological chemistry, 2012Co-Authors: Victoria V Hargreaves, Christopher D Putnam, Richard D. KolodnerAbstract:ATP binding causes the mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces Conformational Changes in Msh2-Msh6; however, the nature of these Conformational Changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a Conformational Change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue Arg(1124). An engineered disulfide bond within this region prevented the ATP-driven Conformational Change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven Conformational Change plays a role in mismatch repair.