Figure 5.

Measurement of the binding affinity between Rev-ARM peptide and RRE, ES-stabilizing mutants and rescue mutants using fluorescence polarization. (A) The designed ES-stabilizing, ES2-stabilizing and ES2-rescue mutants are colored in blue, green and red, respectively. (B-C) Normalized anisotropy values measured for RREIIB (B)and RREII (C) fitted with one-site binding model (see ‘Materials and Methods’ section). The anisotropy value observed in the absence of RNA was normalized to 0.1. Uncertainty reflects the standard deviation from three independent measurements. Buffer conditions: 30 mM HEPES, pH = 7.0, 100 mM KCl, 10mM sodium phosphate, 10 mM ammonium acetate, 10 mM guanidinium chloride, 2 mM MgCl₂, 20 mM NaCl, 0.5 mM EDTA and 0.001% (v/v) Triton-X100. Concentration of Rev-Fl peptide was 10 and 1 nM for RREIIB and RREII respectively.