Figure 5. Analysis of the IFT80 homo-dimer by SEC and SAXS.

(A) SEC analysis (HiLoad Superdex200 column) of various constructs of CrIFT80. Full-length IFT80 elutes at different positions at low and high salt concentration (150 mM and 500 mM NaCl, respectively), indicating that the homo-dimer is salt-labile. This is consistent with the fact that most interactions between the molecules are hydrophilic (see Figure 4). Elution volumes and apparent Mw are indicated for each peak. As SEC is shape-dependent, the Mw values will deviate from the theoretical values. All peaks are far from the void volume of 45 mL for this particular column. The proteolysed IFT80 fragment (residues 1–654) elutes between the low- and high-salt peaks of full-length IFT80, consistent with a dimer, whereas the construct lacking the entire α-solenoid domain (IFT80 BP1-BP2) elutes at significantly lower molecular weight and is monomeric even at low salt concentrations. (B) P(r) vs. radius plot derived from SAXS analysis of full-length IFT80 at low and high salt concentrations (150 mM vs. 500 mM NaCl, respectively), indicating that an IFT80 dimer at low salt is broken up into monomers at high salt. (C) The SAXS envelope calculated for IFT80 at low salt concentrations (grey mesh) provides a much better fit to the crystal structure of the IFT80 homo-dimer (individual molecules shown in cartoon representation in red and blue, respectively), providing proof that IFT80 exists as a homo-dimer in solution at 150 mM NaCl. (D) The SAXS envelope calculated for IFT80 at high salt concentrations (grey mesh) fits better to the crystal structure of a monomeric form of IFT80 (shown in cartoon representation in red), indicating the dimer is broken up into monomers at 500 mM NaCl. Extra density in the SAXS envelope likely corresponds to the C-terminus of IFT80, which is not present in the crystallographic model.