Evolution of a Hydrophobic Core Leads to Fluorescence in Canonical Bacteriophytochromes

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Use of fluorescent proteins to study in vivo processes in mammalian systems begs development of near-infrared (NIR) biomarkers due to superior penetration of the excitation light at longer wavelengths. Bacteriophytochromes (BphPs) that use biliverdin as their chromophore have been engineered to form monomeric NIR biomarkers. Absorption of a red light photon (l=700 nm) leads to isomerization of the C15=C16 bond of BV in the Pr ground state, leading to the second ground state, the far red-light absorbing Pfr. The archetypal NIR phytofluor carried a D207H substitution. The 207 position is important because the main chain carbonyl forms a hydrogen bond with the BV A ring, thus potentially functioning as a proton sink during the photocycle. This variant spurred development of NIR biomarker BPhPs IFP1.4, Wi-Phy, and iRFP. We have solved the structure of IFP1.4 and observe a tightly packed hydrophobic core in the BV binding pocket in contrast to the open pocket in native BphP. Side chains of V173, M174, and V288 form this stabilizing interaction, which is absent in the wild type counterpart because residue 288 is an alanine. We have further disproven the hypothesis that H207 is required for fluorescence, by engineering IFP1.4 D207 and demonstrating it is brighter than any NIR phytofluor described to date. Our research has helped delineate the origin of fluorescence in BphPs and will help phytofluors gain wide spread use as their brightness and resistance to photobleaching are augmented.