Figure 3
A ‘tethered’ CEH-14–LIM-7 complex. (A) Schematic of the CEH-14–LIM-7 tethered complex. Based on data from similar complexes, introducing a tether between the LIM1+2 domains from CEH-14 and the LID from LIM-7 was expected to stabilise the complex and facilitate structural characterisation. This engineering approach takes advantage of the close positioning of C-and N-termini in the native complexes. (B) SEC-MALLS data for CEH-14–LIM-7 in 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT at 0.5 ml/min at 25 °C (black line, refractive index profile; black dots, MW distribution). (C) Far UV-CD profile of CEH-14–LIM-7 (5 μM black dots) in 10 mM Tris-HCl pH 8.5, 150 mM NaF, 0.5 mM TCEP at 20 °C, compared to the spectra of related LHX3/4–ISL1/2 tethered complexes as indicated. Data for those complexes were published previously12. (D) 1H-15N HSQC spectrum of CEH-14–LIM-7 (37 μM) in 20 mM HEPES pH 7.0, 1 mM DTT at 25 °C recorded at 600-MHz.
