EnzyChrom Kit

Two adjacent subunits from this model were selected as the PV N115 2C dimer model. However, the K0.5 value measured was slightly higher than expected. We conclude that RNA binds productively to a 2C dimer and binding of RNA to a 2C monomer may interfere with formation of a 2C dimer capable of achieving the most active catalytically competent state. 3F), it becomes QuantiChrom - EnzyChrom products even clearer that T-antigen uses an entire domain to hold the subunits of the hexamer together. The solvent accessible surface area buried in forming the T-antigen hexamer is on the order of 2600 2, but only 1200 2 for the 2C hexamer. Determinants other than the interactions driven by the carboxy-terminal helix of 2C may be required to form a stable hexamer.
SsRNA 3 and ssRNA 5 were 32P-labeled, indicated by a star. RNA unwinding by 2C was assessed by phosphorimaging of native polyacrylamide gels. HCV NS3 (0.5 M) or WT 2C (2 M) was incubated with 10 nM Duplex 1 or Duplex 2 using two different reaction conditions . The unwinding reactions were quenched after 2, 5, and 15 minutes.

The volume of enzyme added to any reaction was always less than or equal to one-tenth the total reaction volume. Rates are defined as M ADP formed min1 M 2C1. When defining the rate, timepoints were taken during the linear portion of the reaction with less than 20% product formed. Inclusion of 2C in this family makes it likely that 2C would fulfill some type of helicase function, but neither widespread demonstration nor a specific biological target of helicase activity has yet been identified. Similarly, the helicase and/or translocase activity might facilitate encapsidation of viral RNA during morphogenesis. Again, the biological, biochemical, or biophysical evidence to support these assumptions is sparse.
If we assume that heat changes will be undetectable when the protein concentration in the cell reaches a value of 10Kd, 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 l) 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.

HCV NS3 was used as a positive control for RNA unwinding. Buffer indicates quenched reactions without enzyme, and heat indicates reactions heated to 90 C prior to being quenched, resulting in single-stranded RNA. The simplest explanation for these observations is that RNA binds to PV 2C using a two-step mechanism. In the first step, binding is driven by the phosphodiester backbone. In the second step, one or more 2-hydroxyls engage the protein and/or ATP to augment catalysis at the active site. A conserved aspartate of the Walker B motif was restrained to 3.0 from a water molecule of the magnesium coordination sphere and the conserved hydroxyl of the Walker A motif .
The optimized formulation minimizes any interference by substances such as magnesium, lipid, protein and bilirubin. Together, these data suggest that the alpha and beta phosphates are necessary and sufficient for initial binding of nucleotides to 2C. A second step in the binding mechanism would then enable recognition of the base bound and somehow couple the presence of an adenine base to catalytic competence of the active site. Reactions were quenched by the addition of EDTA at a final concentration of 250 mM. During nucleotide competition experiments, the total amount of Mg2+ was stoichiometric compared to the total NTP concentration, with an additional 4 mM Mg2+ free.
RNA-stimulated ATPase activity observed for members of enteroviral species AD. Purified N39 2C proteins (5 g each) from Coxsackievirus B3 , Enterovirus A71 (EV-A71), and Enterovirus D68 (EV-D68) were visualized on a 15% polyacrylamide gel and was stained with Coomassie. RNA-stimulated enteroviral 2C ATPase activity.

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.

The goal of this study was to establish a framework to guide future studies of the specificity, kinetic and chemical mechanisms, and inhibition of the enteroviral 2C ATPase using PV 2C as our model system. This observation is consistent with RNA binding measured by polarization actually reflecting formation of the 2C dimer. Fold stimulation is the quotient of linear rate of RNA-stimulated activity divided by RNA-independent activity. What these experiments do not reveal is whether the dissociation constant measured reflects a value for the 2C dimer, RNA-bound 2C dimer, or some combination thereof. Below, we perform experiments to distinguish between these possibilities. Where X is the concentration of protein, Y is degree of polarization, Kd,app is the apparent dissociation constant, and Ymax is the maximum value of Y.
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 m silica beads, a pore size of 100 Angstrom, and dimensions 7.8 300 mm.