RATIONALLY DESIGNED HELIX-TURN-HELIX PROTEINS AND THEIR CONFORMATIONAL-CHANGES UPON DNA-BINDING

Circular dichroism and electrophoretic mobility shift studies were per formed to confirm that dimerized N-terminal domains of bacterial repre ssors containing helix-turn-helix motifs are capable of high-affinity and specific DNA recognition as opposed to the monomeric N-terminal do mains. Specific, high-affinity DNA binding proteins were designed and produced in which two copies of the N-terminal 1-62 domain of the bact eriophage 434 repressor are connected either in a dyad-symmetric fashi on, with a synthetic linker attached to the C-termini, or as direct se quence repeats. Both molecules bound to their presumptive cognate near ly as tightly as does the natural (full-length and non-covalently dime rized) 434 repressor, showing that covalent dimerization can be used t o greatly enhance the binding activity of individual protein segments, Circular dichroism spectroscopy showed a pronounced increase in the a lpha-helix content when these new proteins interacted with their cogna te DNA and a similar, although 30% lower, increase was also seen upon their interaction with non-cognate DNA. These results imply that a gra dual conformational change may occur when helix-turn-helix motifs bind to DNA, and that a scanning mechanism is just as plausible for this m otif class as that which is proposed for the more flexible basic-leuci ne zipper and basic-helix-loop-helix motifs.