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. 2021 Feb 8;10:e62261. doi: 10.7554/eLife.62261

Figure 4. ORF68 binds nucleic acid in vitro via its central channel.

(a) Electrostatic surface of the ORF68 pentamer, contoured from +5 kT/e (blue) to −5 kT/e (red) and shown from the top (left), bottom (middle), and through the central channel (right). The electrostatic surface lacks regions that were disordered in the structure, including residues 169–172 and 179–188, which face the central channel. The locations of residues selected for mutation are indicated on one monomer of the pentamer. (b) Electrophoretic mobility shift assay using fluorescein-labeled 20 bp dsDNA probe (10 nM) and wild-type or mutant ORF68 (4 μM). (c) Binding curves for wild-type and mutant ORF68 interacting with the 20 bp dsDNA probe were determined by electrophoretic mobility shift assays as in (b). Data represent the mean ± s.d. of three independent experiments. Data were fit with a nonlinear regression to the Hill equation.

Figure 4.

Figure 4—figure supplement 1. ORF68 nonspecifically binds nucleic acid.

Figure 4—figure supplement 1.

(a) Native gel for the electrophoretic mobility shift assay of ORF68 binding to 5′-fluorescein labeled dsDNA probe with 10, 20, or 30 bp. (b) Binding curves for a 85% GC-rich probe (LBS1) and a 50% GC-rich probe (SCRAM) (top). Data represent the mean ± s.d. of three independent experiments. Data were fit with a nonlinear regression to the Hill equation, with best fit derived binding parameters within the 95% CI (bottom): EC50 (effective binding affinity), Bmax (maximum specific binding), H (Hill coefficient), and R2 (goodness of fit).
Figure 4—figure supplement 2. ORF68 mutants can be purified, but mutations in the central channel prevent dsDNA binding.

Figure 4—figure supplement 2.

(a) SDS-PAGE gel of purified recombinant wild-type and mutant ORF68 variants (7.5 μg) used for dsDNA-binding assays. Proteins were visualized by stain-free imaging (top), followed by western blotting for ORF68 (middle), and the Strep tag (bottom). (b) Size exclusion chromatography of wild-type ORF68 and ORF68-K435A reveals that mutation in the central channel does not affect oligomerization. (c) Representative native gels for electrophoretic mobility shift assays with wild-type or mutant ORF68 and a 20 bp dsDNA probe.