The Life of Schmitt 289

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.

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 Å 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 A–D. Purified NΔ39 2C proteins (5 μg each) from Coxsackievirus B3 , Enterovirus A71 (EV-A71), and Enterovirus D68 (EV-D68) were visualized on a 15% polyac