Bacteriophytochrome photoreceptors (BphPs) containing biliverdin (BV) have great potential for the development of genetically engineered near-infrared fluorescent proteins (NIR FPs). We investigated a photoactivatable fluorescent protein PAiRFP1, was engineered through directed molecular evolution. The coexistence of both red light absorbing (Pr) and far-red light absorbing (Pfr) states in dark is essential for the photoactivation of PAiRFP1. The PCR based site-directed reverse mutagenesis, spectroscopic measurements and molecular dynamics (MD) simulations were performed on three targeted sites V386A, V480A and Y498H in PHY domain to explore their potential effects during molecular evolution of PAiRFP1. We found that these substitutions did not affect the coexistence of Pr and Pfr states but led to slight changes in the photophysical parameters. The covalent docking of biliverdin (cis and trans form) with PAiRFP1 was followed by several 100 ns MD simulations to provide some theoretical explanations for the coexistence of Pr and pfr states. The results suggested that experimentally observed coexistence of Pr and Pfr states in both PAiRFP1 and mutants were resulted from the improved stability of Pr state. The use of experimental and computational work provided useful understanding of Pr and Pfr states and the effects of these mutations on the photophysical properties of PAiRFP1.
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