Figure 5.

NMR and SAXS analysis of the effect of tandem serine phosphorylation of SF1. (A) SDS–PAGE analysis of phosphorylation of SF1^NTD and SF1. The phosphorylated protein (red arrow) migrates slower on the gel than the non-phosphorylated. (B) Superposition of ¹H,¹⁵N HSQC NMR spectra of non-phosphorylated (black) and phosphorylated SF1^NTD and SF1 (red). Selected residues, which are shifted upon phosphorylation are annotated. (C) CSPs (red) and residue-specific local correlation times τ_c are shown for SF1^NTD (blue) and pSF1^NTD (red). The secondary structures and the phosphorylation sites are depicted above the diagram. The tandem phosphorylated linker region that rigidifies upon phosphorylation is highlighted by a box. A ribbon representation of SF1^HH colour coded with the phosphorylation-induced CSPs is shown. (D) SAXS data showing comparisons of radial density distributions of non-phosphorylated and phosphorylated SF1^NTD and SF1, in complex with U2AF65^UHM, U2AF65^RRM123 and with U2AF65^RRM123–RNA, respectively.