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. 2015 Sep 18;112(41):E5567–E5574. doi: 10.1073/pnas.1512240112

Fig. S2.

Fig. S2.

(A and B) Size-exclusion chromatograms for dark (OCPO) and illuminated OCP samples applied to a Superdex 200 10/300 GL column in different buffers. Experiments were performed in the following buffers: TBS (50 mM Tris⋅HCl, 200 mM NaCl); PBS (20 mM potassium phosphate, pH 7.4, 100 mM NaCl); 200 mM ammonium acetate buffer. Elution volumes for three globular standards are indicated (vertical lines) on the baseline of each chromatogram. Estimated MWs for each elution peak, as determined from a calibration to the globular standards in each buffer system, are noted on the chromatograms. (C) Circular dichroism spectra of OCPO and OCPR in the far-UV range. The spectra of OCP in darkness (OCPO, black solid line) and after illumination (OCPR, dashed red line) are shown as the average result of four independent experiments (4 × 8 scans per spectrum). Changes in the average mean-residue-ellipticity values between OCPO and OCPR are shown additionally as a difference spectrum (blue dashed line). (D) Far-UV CD difference spectra (OCPR – OCPO) calculated from four independent experiments. Illumination has a small but reproducible effect on the far-UV CD spectra. (E) Time-dependent OCPR CD data collection of OCP samples before and after CD measurements. A representative dataset showing successive scans of the OCPR sample (∼2 min per scan) and the lack of significant thermal reversion to the OCPO form during the full time course of the measurement. An OCPO spectrum (average of eight scans) is also shown for reference. (F) Time-dependent UV-visible (UV-Vis) absorbance spectra of OCP samples before and after CD measurements. UV-Vis spectra collected during the CD experiment: OCPO (black dotted line) was immediately illuminated to OCPR (red solid line) after CD measurements on OCPO. The OCPR spectrum after CD acquisition (dotted red line) was acquired after ∼20 min of spectral averaging (eight scans) at 4 °C in the CD spectrometer. Thermal reversion of OCP after the OCPR measurements resulted in recovery of the OCPO form (black solid line).