EnzyChrom Kit
The column, fraction collector, and autosampler were set to 4 °C. A volume of 15 μl of 2C protein (75 μg) was injected at a flow rate of 0.5 ml min−1 with a chromatogram run time of ∼25 minutes. Data was analyzed using the ASTRA QuantiChrom - EnzyChrom products software from Wyatt. As a single peak with a molar mass corresponding to a dimer of 2C protein was observed by SEC-MALS, BioSAXS data were subsequently collected directly with no further purification via SEC-MALS fractions.
The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here, we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state.
And evaluation of drug effects on calcium metabolism. Quantitative determination of α-amylase activity by colorimetric method. Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion.
If we assume that heat changes will be undetectable when the protein concentration in the cell reaches a value of 10•Kd, then the Kd value for the dimer would be on the order of 400 nM. We will refer to a protein with an authentic amino terminus as WT. Solubility of WT, full-length 2C requires the presence of detergent. The trap strand was a 9-mer RNA of the same sequence as the 32P-labeled strand, 5′-CCGGGCGGC-3′. Reaction timepoints were quenched with an equal volume (5 μl) addition of loading buffer (100 mM ETA, 0.33% (wt/v) SDS, 10% (v/v) glycerol, 0.025% (wt/v) bromophenol blue, 0.025% (wt/v) xylene cyanol). Products were resolved on 20% native PAGE gels by electrophoresis in 1× TBE at 15 mA for 90 min.
Gels were visualized by phosphorimaging. Where kmax is the maximum rate with a substrate concentration of 0.5 mM ATP, C is a constant, and kobs the observed rate (μM ADP min−1 μM 2C−1). Assays can be directly performed on raw biological samples i.e., in the presence of lipid, protein and minerals such as magnesium, iron and zinc. All data are incorporated into the article and its online supplementary material. Constructs and full data sets presented in this study are available upon request to the corresponding author.
Please check for further notifications by email. We are optimistic that the groundwork established here will facilitate elucidation of high-resolution structures of the catalytically competent state. AThe apparent dissociation constant is presented ± standard error with 95% confidence interval provided in brackets below. The procedure involves addition of a single working reagent and incubation for 3 min. Can be readily automated as a high-throughput assay for thousands of samples per day. Your order for QuantiChrom products is prepared and secured by our specialists.
Our QuantiChrom technology and EnzyChrom products will help your company become more competitive.
Binding of RNA and ATP to 2C occurs using a two-step process, which suggests specific interactions between 2C, 2′-hydroxyls of RNA, and both the base and ribose of ATP. These key principles revealed by studying PV 2C protein extend to EV-A71, CVB3, and EV-D68. Is there a biological role for a dimeric, RNA-dependent ATPase during the enteroviral lifecycle? Further studies will be required to address this question. If not, perhaps other scaffolds for may exist to assemble hexamers during genome replication.
2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the α and β phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase. Samples of 2C NΔ39 protein at a concentration of 5 mg min-1were prepared in a buffer of 20 mM HEPES, pH 6.8, 5 mM Magnesium acetate, 1 mM TCEP, 5% glycerol, and 50 mM NaCl. All samples were centrifuged at 6000 ×g for 20 min to minimize aggregation and to remove dust particles prior to SEC-MALS.
Our studies make a compelling case for the existence of a conformation of the PV 2C-ATP complex that links binding of the correct nucleotide to catalytic competence of the active site. As a first step towards identification of other determinants of ATP interrogated in the second step of binding that are required for catalysis, we have investigated the activity of several analogues of ATP. Together, these data suggest that a single binding site exists for binding to single-stranded nucleic acid, and that the primary determinant for binding to this site is the phosphodiester backbone. Use of high protein concentrations is a requirement for this experiment, so it is not possible to glean any insight into the equilibrium dissociation constant for the dimer from this experiment.
SEC chromatographic separation of samples was conducted using an Agilent 1260 Infinity II HPLC system with an autosampler and fraction collector. A Wyatt Technology DAWN MALS and Wyatt Optilab Refractive Index detectors were used for analyzing the molar mass of peaks that eluted from the column. The SEC-MALS system was calibrated with bovine serum albumin and equilibrated with in the same mobile phase as that of the 2C samples. Normalization and alignment of the MALS and RI detectors were carried out using the BSA (monomer ∼66 kDa) standard, run in the 2C buffer condition. The Wyatt SEC hydrophilic column used had 5 μm silica beads, a pore size of 100 Angstrom, and dimensions 7.8 × 300 mm.
The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here, we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state.
And evaluation of drug effects on calcium metabolism. Quantitative determination of α-amylase activity by colorimetric method. Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion.
If we assume that heat changes will be undetectable when the protein concentration in the cell reaches a value of 10•Kd, then the Kd value for the dimer would be on the order of 400 nM. We will refer to a protein with an authentic amino terminus as WT. Solubility of WT, full-length 2C requires the presence of detergent. The trap strand was a 9-mer RNA of the same sequence as the 32P-labeled strand, 5′-CCGGGCGGC-3′. Reaction timepoints were quenched with an equal volume (5 μl) addition of loading buffer (100 mM ETA, 0.33% (wt/v) SDS, 10% (v/v) glycerol, 0.025% (wt/v) bromophenol blue, 0.025% (wt/v) xylene cyanol). Products were resolved on 20% native PAGE gels by electrophoresis in 1× TBE at 15 mA for 90 min.
Gels were visualized by phosphorimaging. Where kmax is the maximum rate with a substrate concentration of 0.5 mM ATP, C is a constant, and kobs the observed rate (μM ADP min−1 μM 2C−1). Assays can be directly performed on raw biological samples i.e., in the presence of lipid, protein and minerals such as magnesium, iron and zinc. All data are incorporated into the article and its online supplementary material. Constructs and full data sets presented in this study are available upon request to the corresponding author.
Please check for further notifications by email. We are optimistic that the groundwork established here will facilitate elucidation of high-resolution structures of the catalytically competent state. AThe apparent dissociation constant is presented ± standard error with 95% confidence interval provided in brackets below. The procedure involves addition of a single working reagent and incubation for 3 min. Can be readily automated as a high-throughput assay for thousands of samples per day. Your order for QuantiChrom products is prepared and secured by our specialists.
Our QuantiChrom technology and EnzyChrom products will help your company become more competitive.
Binding of RNA and ATP to 2C occurs using a two-step process, which suggests specific interactions between 2C, 2′-hydroxyls of RNA, and both the base and ribose of ATP. These key principles revealed by studying PV 2C protein extend to EV-A71, CVB3, and EV-D68. Is there a biological role for a dimeric, RNA-dependent ATPase during the enteroviral lifecycle? Further studies will be required to address this question. If not, perhaps other scaffolds for may exist to assemble hexamers during genome replication.
2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the α and β phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase. Samples of 2C NΔ39 protein at a concentration of 5 mg min-1were prepared in a buffer of 20 mM HEPES, pH 6.8, 5 mM Magnesium acetate, 1 mM TCEP, 5% glycerol, and 50 mM NaCl. All samples were centrifuged at 6000 ×g for 20 min to minimize aggregation and to remove dust particles prior to SEC-MALS.
Our studies make a compelling case for the existence of a conformation of the PV 2C-ATP complex that links binding of the correct nucleotide to catalytic competence of the active site. As a first step towards identification of other determinants of ATP interrogated in the second step of binding that are required for catalysis, we have investigated the activity of several analogues of ATP. Together, these data suggest that a single binding site exists for binding to single-stranded nucleic acid, and that the primary determinant for binding to this site is the phosphodiester backbone. Use of high protein concentrations is a requirement for this experiment, so it is not possible to glean any insight into the equilibrium dissociation constant for the dimer from this experiment.
SEC chromatographic separation of samples was conducted using an Agilent 1260 Infinity II HPLC system with an autosampler and fraction collector. A Wyatt Technology DAWN MALS and Wyatt Optilab Refractive Index detectors were used for analyzing the molar mass of peaks that eluted from the column. The SEC-MALS system was calibrated with bovine serum albumin and equilibrated with in the same mobile phase as that of the 2C samples. Normalization and alignment of the MALS and RI detectors were carried out using the BSA (monomer ∼66 kDa) standard, run in the 2C buffer condition. The Wyatt SEC hydrophilic column used had 5 μm silica beads, a pore size of 100 Angstrom, and dimensions 7.8 × 300 mm.
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