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1.
FIGURE 4.

FIGURE 4. From: Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space.

Oligomeric state of pVI determined by single molecule photo bleaching. A, in an image series (1.2 kW/cm2, 0.00352 s/frame) of a sliding assay of pVI molecules labeled with Cy3B at an efficiency of 0.9 Cy3B molecules/pVI molecule, one of the molecules irreversibly stuck to a spot on the surface of the glass coverslip; the fluorescence intensity at that spot was recorded as a function of time. Bleaching occurred in one step. a.u., arbitrary units. B, an experiment similar to that in A except that a molecule of a (pVIc-biotin)·streptavidin complex labeled with two molecules of Alexa Fluor 5468 stuck to a spot on the surface of the glass coverslip. The bleaching of the (pVIc-biotin)·streptavidin complex occurred in two steps. The red line is the average intensity in that time range.

Vito Graziano, et al. J Biol Chem. 2013 January 18;288(3):2059-2067.
2.
FIGURE 1.

FIGURE 1. From: Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space.

Binding of pVI and VI to DNA. Equilibrium dissociation constants were calculated. Aliquots of pVI or VI were added to buffer B with 1 mm MgCl2 containing 10 nm fluorescein-labeled 33-mer dsDNA, and the steady-state anisotropy after each addition was measured at 21 °C. The data are presented in the form of a Bjerrum plot and yielded apparent equilibrium dissociation constants of 46 ± 1.6 nm for pVI (open circles) and 307 ± 38 nm for VI (open squares). Data points are averages of three replicates ± S.D. The solid lines were obtained by inverse variance weighted nonlinear regression fit of a 1:1 ligand-receptor binding model to the data.

Vito Graziano, et al. J Biol Chem. 2013 January 18;288(3):2059-2067.
3.
FIGURE 2.

FIGURE 2. From: Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space.

Stoichiometry of binding of pVI to DNA. A, stoichiometry of binding. Aliquots of pVI were added to solutions of buffer B containing 13.7 nm 60-bp DNA labeled at one 5′ end with fluorescein. After each addition, the steady-state anisotropy was measured. The solid lines are linear fits to the data. The point of intersection of the straight lines, the dashed vertical line, indicates that a minimum of 110 nm pVI was required to saturate 13.7 nm 60-bp dsDNA, a stoichiometry of binding of 8:1. The experiment was repeated with 33-mer, 18-mer, and 12-mer dsDNA.8 Data points are averages of four replicates ± S.D. B, the number of base pairs occluded upon binding of pVI to DNA. The data from the experiments in A were used to plot the ratio of pVI to DNA versus DNA length (bp). The average number of bp per pVI binding site was 8.

Vito Graziano, et al. J Biol Chem. 2013 January 18;288(3):2059-2067.
4.
FIGURE 6.

FIGURE 6. From: Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space.

Size-exclusion chromatography of hexon·pVI complexes. A, gel filtrations of hexon and hexon·pVI complexes. The samples were injected onto a 7.8 mm × 30-cm TSK-GEL G3000SWXL analytical size-exclusion column and eluted with 25 mm MES, pH 6.5, containing 250 mm NaCl. The vertical marks on the top correspond to elution times of known molecular weight standards, with their molecular masses shown on top. The apparent molecular weights of Ad2 hexon and Ad2 hexon·pVI were determined by interpolation from the standard curve. Solid line, Ad2 hexon; dashed line, Ad2 hexon·pVI complex. B, SDS-polyacrylamide gel electrophoresis of purified proteins VI, pVI, and hexon (lanes 2–4). Lane 1 contains molecular weight markers, with their molecular masses shown on the far left. The proteins were run on a 4–20% polyacrylamide gradient gel and stained with Coomassie Blue.

Vito Graziano, et al. J Biol Chem. 2013 January 18;288(3):2059-2067.
5.
FIGURE 3.

FIGURE 3. From: Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space.

Thermodynamic parameters in the binding of pVI to DNA. A, number of ion pairs involved in binding of pVI to DNA. Binding isotherms of pVI binding to 10 nm fluorescein-labeled 12-mer dsDNA in buffer B are shown as a function of the NaCl concentration (black, 0.02 m; red, 0.03 m; blue, 0.04 m; green, 0.05 m; and pink, 0.06 m). Binding was measured by changes in fluorescence anisotropy as the pVI concentration was increased, as described under “Experimental Procedures.” Data points are averages of four replicates ± S.D. The solid curves are inverse weighted nonlinear regression fit of the data to a 1:1 binding model. B, changes in nonelectrostatic free energy upon the binding of pVI to DNA. The log of the equilibrium dissociation constants calculated from the data in A are plotted versus −log [NaCl]. The error bars denote the S.E. in the Kd value obtained from the nonlinear regression fit of the data in A. The solid line is a weighted linear regression fit of the data.

Vito Graziano, et al. J Biol Chem. 2013 January 18;288(3):2059-2067.
6.
FIGURE 5.

FIGURE 5. From: Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space.

Equilibrium binding and tight binding of pVI to hexon. A, change in fluorescence intensity when 1 nm Cy3B-pVI is titrated with hexon in 20 mm Hepes, 150 mm NaCl, pH 7. Both pVI and hexon concentrations are reported as monomer concentrations. The dissociation constant (Kd) was determined by inverse variance weighted nonlinear regression fit of the steady-state fluorescence intensity data to a single site receptor-ligand binding model. The Kd is 1.1 ± 0.16 nm. Data points are averages of five replicates ± S.D. B, binding data for pVI-hexon interaction under tight binding condition. Changes in fluorescence anisotropy are observed when 20 nm of Cy3B-pVI is titrated with increasing amounts of hexon (monomer concentration) in buffer containing 20 mm Hepes, 150 mm NaCl, pH 7. The stoichiometry of binding is determined by the intersection of the two linear regression lines from the first seven and last six data points. Data points are averages of four replicates ± S.D.

Vito Graziano, et al. J Biol Chem. 2013 January 18;288(3):2059-2067.

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