Figure 3. The liquid-like phase formed by rpL5 and N130 is characterized by molecular ordering and is accompanied by soluble, oligomeric intermediates.
(a) Fluorescence anisotropy of Alexa Fluor594-labeled N130 (at S125C within acidic tract A2; 1 μM) upon titration of the rpL5 peptide; the total [N130] was 200 μM. Insets: light microscopy images of the 1:1 (cyan box), 2:1 (green box) and 3:1 (red box) rpL5:N130 solutions. The same stoichiometry color coding is used in all panels. (b) SANS curves, I(q) versus q, for rpL5:N130 (200 μM) solutions at 0:1, 1:1, 2:1, and 3:1 stoichiometry. The curve for the 0:1 solution is on the absolute I(q) scale (cm-1) with the others shifted in 1, 2 and 4 decade increments for clarity. Fits (solid lines) of the curves to obtain Rg values (0:1 and 1:1 solutions) and correlation distances (2:1 and 3:1 solutions) are shown (See Analysis methods for details on curve fitting). (c) Pair-distribution, P(r), curves for 0:1 and 1:1 rpL5:N130 were calculated from the corresponding SANS curves (fits shown in Figure 3b). For apo N130, the Dmax ~ 78.65 Å with a resulting Rg = 23.04 ± 0.09 Å and I(0) = 0.2344 ± 0.0006 cm-1. From I(0) and using Eq. S1, the estimated molecular mass, M = 76 kDa, was determined; this mass is consistent with the expected five subunits within the pentamer (subunit M = 14.6 kDa). For 1: 1 rpL5:N130, the Dmax ~ 80.32 Å with a resulting Rg = 24.3 ± 0.1 Å and I(0) = 0.2744 ± 0.0009 cm-1. Here, I(0) yields M = 89 kDa, indicative of ~5 rpL5 (M = 2.2 kDa) molecules bound to N130.
Figure 3—figure supplement 1. Sedimentation velocity analytical ultracentrifugation (SV-AUC) profiles for N130 titrated with rpL5.
