Curated Optogenetic Publication Database

Abstract: Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Abstract: Phytochromes are biological photoreceptors found in all kingdoms of life. Numerous physicochemical and spectroscopic studies of phytochromes have been carried out for many decades, both experimentally and computationally, with the main focus on the photoconversion mechanism involving a tetrapyrrole chromophore. In this computational work, we concentrate on the long-scale dynamic motion of the photosensory domain of Deinococcus radiodurans by means of classical all-atom molecular dynamics (MD) simulations. Conventional and accelerated MD methods in combination with two different force fields, CHARMM27 and AMBER ff14SB, are tested in long atomistic simulations to confront the dynamics of monomer and dimer forms. These calculations highlight dissimilar equilibrium conformations in aqueous solutions and, in turn, different large-scale dynamic behaviors of the monomer form vs the dimer form. While the phytochrome in a monomer form tends to close the cavity entailed between the GAF and PHY domains, the opposite trend is predicted for the phytochrome dimer, which opens up as a consequence of the formation of strong salt bridges between the PHY domains of two molecules in water.