Kinesin Eg5 is a mitotic motor protein essential for bipolar spindle formation during cell division. To achieve light-based control of Eg5 activity, we engineered fusion proteins incorporating two light-responsive domains: Photozipper (PZ) and Dronpa 145N. PZ, a blue-light receptor derived from aureochrome 1, contains a leucine zipper, basic region, and LOV domain, and undergoes light-induced dimerization that enhances DNA-binding affinity. Three PZ-fusion constructs were generated: (1) wild-type Eg5-PZ, (2) truncated Eg5-PZ, and (3) PZ-Eg5, all with equal spacer lengths and expressed in E. coli . Separately, Eg5 was fused to Dronpa 145N, a fluorescent protein that switches from a non-fluorescent monomer to a fluorescent tetramer upon specific light exposure. Spectroscopy and chromatography confirmed that PZ fusions underwent light-induced dimerization, whereas Dronpa 145N fusions showed no significant isomerization changes. The zipper domain stabilized the monomer in the dark, while the LOV domain and zipper-zipper interactions promoted dimerization under blue light. Functional assays demonstrated that Eg5 ATPase activity and motility were regulated by blue light through PZ-mediated conformational changes in the presence of microtubules. In contrast, Dronpa 145N did not confer light-dependent modulation of Eg5 activity. These results indicate that Photozipper enables reversible, light-dependent control of Eg5 function, providing a promising tool for optogenetic manipulation of motor proteins. Although Dronpa 145N was ineffective in this context, our system underscores the potential of light-regulated proteins in cell biology and therapeutic research.
Stanley Tabi Besong (Sun,) studied this question.