Equilibrium Mg2+- and Mn2+-induced folding of unmodified P4–P6 (A) and phosphorothioate-substituted variants (B–F) in a background of 2 M NaCl (with 50 mM Na-MOPS at pH 7). Mg2+ data points are shown as open symbols and Mn2+ data points are shown as closed symbols. Values of the fractional protection from hydroxyl radical cleavage derived from two to four independent experiments for subdomain nucleotides 164 (triangles), 176–177 (circles), 180–182 (squares), and 187 (diamonds) were averaged according to the procedure outlined in the Materials and Methods and Supplemental Material. The resulting values were fit to the Hill equation to yield curves whose midpoints and Hill coefficients reflect the appearance of protections over all of the protected nucleotides simultaneously. Data for nucleotide 187 are not included for certain variants due to nonspecific degradation of the phosphorothioate linkage, which interfered with the protection signal at this site.
