| Summary: | The Cro family of bacteriophage DNA-binding proteins demonstrates the substantial conformational changes that can occur in protein evolution while the primary sequence is significantly conserved. Xfaso 1 and Pfl 6 of the Cro family are -helical and mixed -helical/- sheet, respectively, despite sharing 40% sequence identity. Both proteins bind DNA using a helix-turn-helix (HTH) motif, however, the natural consensus DNA sequences of the proteins are different at three positions in each seven base-pair half site. Fluorescence anisotropy measurements showed that wild-type Xfaso 1 and Pfl 6 bound their cognate sites with dissociation constants (K(d)) of 230 nM and 56 nM, respectively. Wild-type Pfl 6 bound its noncognate site with K(d) = 1.99 μM and wild-type Xfaso 1 did not bind its noncognate site. We introduced mutations into the HTH region of both proteins in order to equalize the binding region sequence while retaining global structure. By exchanging the HTH sequence of the two proteins the specificity of binding was switched from cognate to noncognate consensus site. We found that the local sequence is the primary determinant in the DNA binding specificity for Xfaso 1 and Pfl 6, and the global conformation is not the major difference in binding specificity.
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