Structure-guided Engineering Enhances a Phytochrome-based Infrared Fluorescent Protein

Phytochrome is a multidomain dimeric red light photoreceptor that utilizes a chromophore-binding domain (CBD), a PHY domain, and an output module to induce cellular changes in response to light. A promising biotechnology tool emerged when a structure-based substitution at Asp-207 was shown to be an infrared fluorophore that uses a biologically available tetrapyrrole chromophore. We report multiple crystal structures of this D207H variant of the Deinococcus radiodurans CBD, in which His-207 is observed to form a hydrogen bond with either the tetrapyrrole A-ring oxygen or the Tyr-263 hydroxyl. Based on the implications of this duality for fluorescence properties, Y263F was introduced and shown to have stronger fluorescence than the original D207H template. Our structures are consistent with the model that the Y263F change prevents a red light-induced far-red light absorbing phytochrome chromophore configuration. With the goal of decreasing size and thereby facilitating use as a fluorescent tag in vivo, we also engineered a monomeric form of the CBD. Unexpectedly, photoconversion was observed in the monomer despite the lack of a PHY domain. This observation underscores an interplay between dimerization and the photochemical properties of phytochrome and suggests that the monomeric CBD could be used for further studies of the photocycle. The D207H substitution on its own in the monomer did not result in fluorescence, whereas Y263F did. Combined, the D207H and Y263F substitutions in the monomeric CBD lead to the brightest of our variants, designated Wisconsin infrared phytofluor (Wi-Phy).