Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:M... more Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome, controlling transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers to determine TF effect on chromatin architecture and shed light on its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by...
Human peroxiredoxin 6 (PRDX6) is the only mammalian 1-Cys based member of the Prx family.1 In the... more Human peroxiredoxin 6 (PRDX6) is the only mammalian 1-Cys based member of the Prx family.1 In the first step of its catalytic cycle a sulfenic acid (CP-SOH) is formed at the peroxidatic cysteine, C47, which is then reduced by a thiol-containing agent like glutathione or by ascorbate. The finding of ascorbate as a Prx reducing agent2 revolutionized the thiol-specific antioxidant paradigm, expanding the world of possible 1-Cys Prxs reductants. Rate constants for reduction of CP-SOH by ascorbate have been measured for different 1-Cys based Prxs at L. Netto’s Lab, obtaining values in the 500-2000 M-1s-1 range. Here we address the molecular mechanism of PRDX6 CP-SOH reduction by ascorbate from a theoretical perspective, by combining molecular dynamics and ligand-protein docking with QM/MM methods. Our results show the reaction follows a SN2-like substitution mechanism coupled at the transition state with a proton transfer (PT) from R155 into the leaving OH-. A free-energy activation barr...
ABSTRACT Human peroxiredoxin-5 (PRDX5) is a thiol peroxidase that reduces H2O2 10(5) times faster... more ABSTRACT Human peroxiredoxin-5 (PRDX5) is a thiol peroxidase that reduces H2O2 10(5) times faster than free cysteine. To assess the influence of two conserved residues on the reactivity of the critical cysteine (C47), we determined the reaction rate constants of PRDX5, wild type (WT), T44V and R127Q with one substrate electrophile (H2O2) and a nonspecific electrophile (monobromobimane). We also studied the corresponding reactions of low molecular-weight thiolates (LMWT) in order to construct a framework against which we could compare our proteins. To obtain a detailed analysis of the structural and energetic changes involved in the reaction between WT PRDX5 and H2O2, we performed ONIOM QM/MM calculations with a QM region including 60 atoms of substrate and active-site described by the B3LYP density functional and the 6 31+G(d,p) basis set; the rest of the protein was included in the MM region. Brønsted correlations reveal that the absence of T44 can increase the general nucleophilicity of the C47, but decrease the specific reactivity towards H2O2 by a factor of 10(3). R127Q mutation causes C47 to behave like a LMWT in the two studied reactions. QM/MM results with WT PRDX5 showed that hydrogen bonds in the active site are the cornerstone of two effects that make catalysis possible: the enhancement of thiolate nucleophilicity upon substrate ingress and the stabilization of the transition state. In both effects T44 has a central role. These effects occur in a precise temporal sequence that ensures the selective nucleophilicity of C47 is available only for peroxide substrates.
A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) h... more A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) has been developed to measure nucleophilicity and acidity of protein and low-molecular-weight thiols. Nucleophilicity of the thiolate is measured as the pH-independent second-order rate constant of its reaction with mBBr. The ionization constants of the thiols are obtained through the pH dependence of either second-order rate constant or initial rate of reaction. For readily available thiols, the apparent second-order rate constant is measured at different pHs and then plotted and fitted to an appropriate pH function describing the observed number of ionization equilibria. For less available thiols, such as protein thiols, the initial rate of reaction is determined in a wide range of pHs and fitted to the appropriate pH function. The method presented here shows excellent sensitivity, allowing the use of nanomolar concentrations of reagents. The method is suitable for scaling and high-throughput screening. Example determinations of nucleophilicity and pK(a) are presented for captopril and cysteine as low-molecular-weight thiols and for human peroxiredoxin 5 and Trypanosoma brucei monothiol glutaredoxin 1 as protein thiols.
Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:M... more Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome, controlling transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers to determine TF effect on chromatin architecture and shed light on its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by...
Human peroxiredoxin 6 (PRDX6) is the only mammalian 1-Cys based member of the Prx family.1 In the... more Human peroxiredoxin 6 (PRDX6) is the only mammalian 1-Cys based member of the Prx family.1 In the first step of its catalytic cycle a sulfenic acid (CP-SOH) is formed at the peroxidatic cysteine, C47, which is then reduced by a thiol-containing agent like glutathione or by ascorbate. The finding of ascorbate as a Prx reducing agent2 revolutionized the thiol-specific antioxidant paradigm, expanding the world of possible 1-Cys Prxs reductants. Rate constants for reduction of CP-SOH by ascorbate have been measured for different 1-Cys based Prxs at L. Netto’s Lab, obtaining values in the 500-2000 M-1s-1 range. Here we address the molecular mechanism of PRDX6 CP-SOH reduction by ascorbate from a theoretical perspective, by combining molecular dynamics and ligand-protein docking with QM/MM methods. Our results show the reaction follows a SN2-like substitution mechanism coupled at the transition state with a proton transfer (PT) from R155 into the leaving OH-. A free-energy activation barr...
ABSTRACT Human peroxiredoxin-5 (PRDX5) is a thiol peroxidase that reduces H2O2 10(5) times faster... more ABSTRACT Human peroxiredoxin-5 (PRDX5) is a thiol peroxidase that reduces H2O2 10(5) times faster than free cysteine. To assess the influence of two conserved residues on the reactivity of the critical cysteine (C47), we determined the reaction rate constants of PRDX5, wild type (WT), T44V and R127Q with one substrate electrophile (H2O2) and a nonspecific electrophile (monobromobimane). We also studied the corresponding reactions of low molecular-weight thiolates (LMWT) in order to construct a framework against which we could compare our proteins. To obtain a detailed analysis of the structural and energetic changes involved in the reaction between WT PRDX5 and H2O2, we performed ONIOM QM/MM calculations with a QM region including 60 atoms of substrate and active-site described by the B3LYP density functional and the 6 31+G(d,p) basis set; the rest of the protein was included in the MM region. Brønsted correlations reveal that the absence of T44 can increase the general nucleophilicity of the C47, but decrease the specific reactivity towards H2O2 by a factor of 10(3). R127Q mutation causes C47 to behave like a LMWT in the two studied reactions. QM/MM results with WT PRDX5 showed that hydrogen bonds in the active site are the cornerstone of two effects that make catalysis possible: the enhancement of thiolate nucleophilicity upon substrate ingress and the stabilization of the transition state. In both effects T44 has a central role. These effects occur in a precise temporal sequence that ensures the selective nucleophilicity of C47 is available only for peroxide substrates.
A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) h... more A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) has been developed to measure nucleophilicity and acidity of protein and low-molecular-weight thiols. Nucleophilicity of the thiolate is measured as the pH-independent second-order rate constant of its reaction with mBBr. The ionization constants of the thiols are obtained through the pH dependence of either second-order rate constant or initial rate of reaction. For readily available thiols, the apparent second-order rate constant is measured at different pHs and then plotted and fitted to an appropriate pH function describing the observed number of ionization equilibria. For less available thiols, such as protein thiols, the initial rate of reaction is determined in a wide range of pHs and fitted to the appropriate pH function. The method presented here shows excellent sensitivity, allowing the use of nanomolar concentrations of reagents. The method is suitable for scaling and high-throughput screening. Example determinations of nucleophilicity and pK(a) are presented for captopril and cysteine as low-molecular-weight thiols and for human peroxiredoxin 5 and Trypanosoma brucei monothiol glutaredoxin 1 as protein thiols.
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