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. Author manuscript; available in PMC: 2010 Feb 19.

Published in final edited form as: J Biol Chem. 2006 Sep 27;281(47):35802–35811. doi: 10.1074/jbc.M608303200

Fig. 1 — A. The pH-dependent conformational alteration of soluble Bcl-2ΔTM was assessed by monitoring the tryptophan fluorescence intensity in solutions of various pH. Data shown are averages of relative intensities (F/F₀) from three independent measurements (○) with standard deviations (S.D.; error bars). F₀, the intensity measured at pH 7.4; F, the intensity measured at other pH. B. Solvent exposure of the hydrophobic helix 5 of Bcl-2ΔTM was assessed by measuring I⁻ quenching of fluorescence intensity of an NBD dye attached to Cys¹⁵⁸ in helix 5. F₀ is the net fluorescence intensity prior to addition of KI, and F the dilution-corrected fluorescence intensities after addition of indicated concentrations of KI. Data shown are averages of fluorescence quenching (F₀/F-1) from two independent experiments conducted either at pH 7.4 (○) or 5.0 (△) with S.D. (error bars). The linear least-squares best-fit lines are also shown. C. Bcl-2ΔTM homo-binding in solution was monitored by measuring the tryptophan fluorescence anisotropy (r_W) at various protein concentrations. Data shown are averages from three independent titrations conducted either at pH 7.4 (○) or 5.0 (△) with S.D. (error bars) and nonlinear least-squares best-fit curves (dash or solid) that were generated using the quadratic equation as described(31). D. Binding of Bax H2-H3 peptide to Bcl-2ΔTM at pH 5.0 was monitored by measuring the CF fluorescence anisotropy (r_CF) of a CF-labeled Bax H2 peptide after titrating Bcl-2ΔTM. Data shown are averages from three independent titrations with S.D. (error bars), conducted either in the absence (□) or presence of an unlabeled Bax H2-H3 peptide (○) or its mutant (G67R) (△). Also shown are the nonlinear least-squares best-fit curves for the former two data sets that were generated using the cubic and quartic equations, respectively, as described(31).

Fig. 1 — A. The pH-dependent conformational alteration of soluble Bcl-2ΔTM was assessed by monitoring the tryptophan fluorescence intensity in solutions of various pH. Data shown are averages of relative intensities (F/F₀) from three independent measurements (○) with standard deviations (S.D.; error bars). F₀, the intensity measured at pH 7.4; F, the intensity measured at other pH. B. Solvent exposure of the hydrophobic helix 5 of Bcl-2ΔTM was assessed by measuring I⁻ quenching of fluorescence intensity of an NBD dye attached to Cys¹⁵⁸ in helix 5. F₀ is the net fluorescence intensity prior to addition of KI, and F the dilution-corrected fluorescence intensities after addition of indicated concentrations of KI. Data shown are averages of fluorescence quenching (F₀/F-1) from two independent experiments conducted either at pH 7.4 (○) or 5.0 (△) with S.D. (error bars). The linear least-squares best-fit lines are also shown. C. Bcl-2ΔTM homo-binding in solution was monitored by measuring the tryptophan fluorescence anisotropy (r_W) at various protein concentrations. Data shown are averages from three independent titrations conducted either at pH 7.4 (○) or 5.0 (△) with S.D. (error bars) and nonlinear least-squares best-fit curves (dash or solid) that were generated using the quadratic equation as described(31). D. Binding of Bax H2-H3 peptide to Bcl-2ΔTM at pH 5.0 was monitored by measuring the CF fluorescence anisotropy (r_CF) of a CF-labeled Bax H2 peptide after titrating Bcl-2ΔTM. Data shown are averages from three independent titrations with S.D. (error bars), conducted either in the absence (□) or presence of an unlabeled Bax H2-H3 peptide (○) or its mutant (G67R) (△). Also shown are the nonlinear least-squares best-fit curves for the former two data sets that were generated using the cubic and quartic equations, respectively, as described(31).