Photo-control of DNA binding by an engrailed homeodomain-photoactive yellow protein hybrid

A photo-controlled version of the engrailed homeodomain (zENG) was created by inserting the homeodomain into a surface loop of a circularly permuted version of the photoactive yellow protein (cPYP). The two proteins fold independently as judged by NMR and fluorescence denaturation measurements. In the dark, the affinity of the zENG domain for its cognate DNA is inhibited >100-fold compared to wild-type zENG. Blue-light irradiation of the hybrid protein leads to enhanced conformational dynamics of the cPYP portion and a two-fold enhancement of the DNA binding affinity of the zENG domain. These results suggest that insertion into a surface loop of cPYP can be a general approach for conferring an initial level of photo-control on a given target protein. Focussed mutation/selection strategies may then be used to enhance the degree of photo-control.