Macromolecular crowding effect for photoreactions of LOV2 domains of Arabidopsis thaliana phototropin 1

Abstract

Phototropins (phot1 and phot2) are blue light sensors consisting of two N-terminal LOV (light–oxygen–voltage sensing) domains (LOV1 and LOV2), a serine/threonine kinase domain at the C-terminus, and a linker region connecting these domains. The chemical reactions of the phot1LOV2 domain and phot1LOV2-linker domain have been studied mostly in buffer solutions. In this study, crowding effects on photoreactions of phot1LOV2 and phot1LOV2-linker constructs were investigated. The conformations of both samples in the dark states monitored by ultraviolet/visible absorption and circular dichroism spectra were not influenced by the presence of the crowding agent Ficoll-70. Conversely, with increasing concentrations of Ficoll-70, the transient grating signal representing the dissociation reaction of the phot1LOV2 dimer was observed to increase, and the dimerization reaction of the phot1LOV2 monomer decreased. This effect was interpreted in terms of an equilibrium shift from the monomer to the dimer in the dark under crowding conditions. The time-dependent circular dichroism signal indicated that the reaction of phot1LOV2-linker remained unchanged in the presence of the crowding reagent, i.e., the linker is unfolded upon photoexcitation. However, the reaction rate constant decreased slightly with increasing concentration of Ficoll-70. Although the viscosity increased ~10-fold at a Ficoll-70 concentration of 200g/L, the rate constant decreased only by 1.5-fold. This apparent insensitivity to crowding effects is explained by local structural changes to the linker that are not obstructed by crowding agents such as Ficoll-70.