Skip to main content
. Author manuscript; available in PMC: 2014 Jan 22.
Published in final edited form as: Biochemistry. 2013 Jan 11;52(3):520–536. doi: 10.1021/bi301074r

Figure 4. Biophysical characterization of the unphosphorylated hSLBP-hSLIP1 complex.

Figure 4

(A) Gel filtration profile of the hSLBP-hSLIP1 complex co-purified in the bacterial expression system after nickel affinity chromatography as the initial step. The SDS-PAGE of the corresponding fractions is shown. The apparent molecular weight of SLBP on a 4–20% SDS-PAGE gradient gel is ~37 kDa and that of hSLIP1 is 26 kDa. The presence of hSLBP and hSLIP1 was confirmed by western blotting using antibodies specific for the two proteins. hSLBP is present only in the first peak corresponding to the complex, whereas hSLIP1 is present in all fractions. The final concentrated sample of the complex shows a stoichiometric amount of hSLBP and hSLIP1. (B) Circular dichroism (CD) spectra of 5 µM free SLIP1 dimer (in blue) and the HSLBP-hSLIP1 complex (in red) is shown. The CD spectrum of free SLIP1 dimer shows a double minimum at 209 and 222 nm that is typical of helical secondary structure in proteins. There is an increase in helical signal in the complex that could correspond to increased helical character of hSLBP in the complex. (C) Gel retardation experiment (EMSA) comparing the binding of full length baculovirus expressed SLBP (lane 2), SLBP RNA binding and processing domain (lane 3) and the bacterially expressed and purified hSLBP-hSLIP1 complex (lane 4) shown in (A). The free probe that is 32P-labeled at the 5’ end is shown in lane 1. (D) Sedimentation velocity profiles of the hSLIP1-hSLBP complex shown in (A) at four different concentrations (0.20 mg/mL (red), 0.10 mg/mL (blue), 0.03 mg/mL (green), and 0.01 mg/mL (black). No concentration dependent change in the normalized sedimentation patterns was observed, demonstrating that there is no dissociation of the complex over this concentration range. (E) Sedimentation velocity profiles of the free histone stem-loop RNA (black), hSLIP-hSLBP complex (blue), hSLIP1-hSLBP complex + 2 molar excess of histone stem-loop RNA (green), and the renormalized sum of the curves for free RNA and for hSLIP1-hSLBP complex (red), demonstrating that there is no interaction between the RNA and the complex under these conditions.