Small GTPase Ras fused with light responsive protein exhibits photo-reversible regulation of GTPase activity.

Abstract

The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing conformational changes in nucleotide-binding motifs have been well studied at the molecular level. Previously, we have demonstrated that H-Ras GTPase cycle activity is photoreversibly regulated by photochromic H-Ras, which is modified with 4-chloroacetoamido-4-sulfo-azobenzene (CASAB). However, the efficiency of photoregulation was not sufficient to utilize as a photo-switching. In addition, direct chemical modification to H-Ras carries risk of inactivating Ras function and chemical modification of H-Ras is hard to apply in vivo experiment. In this study, we tried to photoregulate H-Ras activity using photochromic protein; Dronpa or Aureochrome1. We designed H-Ras fusion Dronpa (HRas-Dronpa) or Aureochrome1 (HRas-Aureo1) and the recombinant proteins were prepared by E. coli expression system. HRas-Dronpa showed photo-isomerization upon 366-nm and 500-nm light irradiations. However, multimer formation accompanied by photoisomerization was not observed. On the other hand, HRas-Aureo1 also showed photo-isomerization accompanied by blue light irradiation and in the dark, and HRas-Aureo1 exhibited dimerization by light irradiation. Moreover, GTPase activity of HRas-Dronpa was changed accompanied by isomerization. GTPase activity of HRas-Aureo1 in the dark was approximately 50% of that under blue light irradiation. It has been suggested that light responsive protein is applicable to photo switching of enzymatic activity for small GTPase.