Figure S4. Biophysical characterization... | Rockefeller University Press

Figure S4.

$Biophysical characterization of CPAP-CC1, CP110-CC2, and CPAP-CC1/CP110-CC2 complex. (A) SEC-MALS analyses of CPAP-CC1 (magenta lines) and CP110-CC2 (green lines) alone, and mixtures of CPAP-CC1 with CP110-CC2 at molar ratios of 1:1 (black line), 2:1 (light blue line), and 3:1 (dark blue line). (B) Coomassie-stained SDS-PAGE of the fractions F1–F5 indicated in panel A and collected from SEC-MALS runs obtained with mixtures of CPAP-CC1 and CP110-CC2. SDS-PAGE analysis of the elution peak fractions centered at around 14.3 ml (corresponding to the molecular weight of CPAP-CC1/CP110-CC2 heterodimer) of the various mixtures revealed equally intense protein bands corresponding to CPAP-CC1 and CP110-CC2. (C and D) SAXS analysis of the CP110-CC2 homodimer. (C) Solution X-ray scattering intensity over scattering angle from CP110-CC2. The fit to the data yielding the interatomic distance distribution is shown with a black line. (D) Surface representation of the X-ray scattering volume of CP110-CC2, at 30 ± 2 Å estimated precision, derived from averaging 22 particle models calculated by ab initio fit to the scattering data. (E) Table summarizing biophysical parameters of CPAP-CC1, CP110-CC2, and an equimolar mixture of CPAP-CC1 and CP110-CC2 obtained by SEC-MALS, CD, and SAXS. (F and G) CD spectrum (F) recorded at 15°C and thermal-unfolding profiles (G) recorded by CD at 222 nm of CP110-CC2 R656A/L659A (light green dashed lines). Source data are available for this figure: SourceData FS4.$

Biophysical characterization of CPAP-CC1, CP110-CC2, and CPAP-CC1/CP110-CC2 complex. (A) SEC-MALS analyses of CPAP-CC1 (magenta lines) and CP110-CC2 (green lines) alone, and mixtures of CPAP-CC1 with CP110-CC2 at molar ratios of 1:1 (black line), 2:1 (light blue line), and 3:1 (dark blue line). (B) Coomassie-stained SDS-PAGE of the fractions F1–F5 indicated in panel A and collected from SEC-MALS runs obtained with mixtures of CPAP-CC1 and CP110-CC2. SDS-PAGE analysis of the elution peak fractions centered at around 14.3 ml (corresponding to the molecular weight of CPAP-CC1/CP110-CC2 heterodimer) of the various mixtures revealed equally intense protein bands corresponding to CPAP-CC1 and CP110-CC2. (C and D) SAXS analysis of the CP110-CC2 homodimer. (C) Solution X-ray scattering intensity over scattering angle from CP110-CC2. The fit to the data yielding the interatomic distance distribution is shown with a black line. (D) Surface representation of the X-ray scattering volume of CP110-CC2, at 30 ± 2 Å estimated precision, derived from averaging 22 particle models calculated by ab initio fit to the scattering data. (E) Table summarizing biophysical parameters of CPAP-CC1, CP110-CC2, and an equimolar mixture of CPAP-CC1 and CP110-CC2 obtained by SEC-MALS, CD, and SAXS. (F and G) CD spectrum (F) recorded at 15°C and thermal-unfolding profiles (G) recorded by CD at 222 nm of CP110-CC2 R656A/L659A (light green dashed lines). Source data are available for this figure: SourceData FS4.