Figure 1. SAS-5 comprises two independently folded domains.

(A) Schematic representation of SAS-5 architecture showing the relative locations and residue boundaries of the coiled-coil and Implico domains, the putative SAS-4 binding site (Hatzopoulos et al., 2013) and the SAS-6 binding site (Qiao et al., 2012; Hilbert et al., 2013). (B) Overlaid CD spectra of SAS-5_Δ282–295 and SAS-5_FLEX samples recorded at 10°C, shown as per residue molar elipticity vs wavelength. The semi-quantitative contribution of secondary structure elements in each spectrum is deconvoluted in the bar charts on the left. Grey color corresponds to random coil, green to β-strand and red to α-helical segments. (C) Thermal unfolding profiles of the same samples monitored by recording molar elipticity at 222 nm as a function of temperature, and graphical representation of the melting transition temperatures observed in each sample. (D–G) Similar CD spectra and thermal unfolding profiles of (D,E) SAS-5_CC and SAS-5_Imp, and (F,G) SAS-5_125–265 samples.

DOI: http://dx.doi.org/10.7554/eLife.07410.003

Figure 1—figure supplement 1. Recombinant proteins sample quality.

(A,B) Sections of SDS-PAGE showing Coomassie-stained samples of recombinant proteins used in this study. Panel A groups proteins with molecular weight over 30 kDa; panel B shows smaller proteins. Individual gel sections have been compressed or expanded along the vertical axis to match the molecular weight marker, and their contrast levels have been equalized.

DOI: http://dx.doi.org/10.7554/eLife.07410.004

Figure 1—figure supplement 2. SAS-5 forms protein aggregates in vitro.

(A) CD spectrum of SAS-5_FL at 10°C shown as per residue molar elipticity against wavelength, and (B) thermal unfolding profile of the same protein monitored by recording molar elipticity at 222 nm as a function of temperature. The CD spectrum is highly similar to that of protein aggregates characterized elsewhere (Digambaranath et al., 2010). (C) Overlay of SEC-MALS chromatograms of SAS-5_FL samples at multiple concentrations, showing scaled light scattering intensity vs elution volume. (D) Representative negative stain electron micrograph of SAS-5_FL and high-magnification view of the boxed section.

DOI: http://dx.doi.org/10.7554/eLife.07410.005

Figure 1—figure supplement 3. A short SAS-5 segment promotes protein aggregation.

(A, B) Coomassie-stained SDS-PAGE of metal-affinity purified SAS-5 variants, corresponding to MsyB-tagged full-length protein and C-terminal truncations (A). The expected SAS-5-MsyB protein bands are indicated by black arrows. Soluble protein yields increased significantly upon truncation of residues 280–296 (compare lanes 3 and 4). Excision of this protein segment (SAS-5_Δ282–295-MsyB) or substitution by a flexible linker (SAS-5_FLEX-MsyB) improved soluble yields of the full-length protein (B, compare to right-most lane of panel A).

DOI: http://dx.doi.org/10.7554/eLife.07410.006

Figure 1—figure supplement 4. SAS-5 secondary structure and disorder predictions.

(A) Schematic representation and relative locations of secondary structure elements predicted from the SAS-5 amino acid sequence by PSIPRED (Jones, 1999). (B) Disorder probability per amino acid residue predicted from the SAS-5 sequence by DISOPRED3 (Jones and Cozzetto, 2014).

DOI: http://dx.doi.org/10.7554/eLife.07410.007

Figure 1—figure supplement 5. The SAS-5 N- and C-terminal segments are unstructured in isolation.

(A) Overlaid CD spectra of SAS-5 N-terminal (residues 2–122) and C-terminal (residues 269–404) fragments recorded at 10°C. The semi-quantitative contribution of secondary structure elements in each spectrum is deconvoluted in the bar chart left. Grey colour corresponds to random coil, green to β-strand and red to α-helical segments. (B) Thermal unfolding profiles of the same samples based on their CD signal at 222 nm.

DOI: http://dx.doi.org/10.7554/eLife.07410.008