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.
Highlights
Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo
First we studied the BV incorporation dynamics of the miRFP670/ C20A mutant that has a single Cys residue in the GAF domain (Fig. 1)
In BphP1-FP, which is a dimeric protein engineered from the same parental protein as miRFP670 (RpBphP1)[23], C–S binding to Cys in the GAF domain both via C31 and C32 were detected in its 3D structure
Summary
Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. The brightness of engineered NIR FPs in mammalian cells and tissues is a complex function of their intrinsic fluorescence properties (molecular brightness), the efficiency of BV binding and the presence of other tetrapyrroles. A key quality of our recently developed watermarked stimulated Raman method[16,17,18,19,20,21,22] is that high-quality ground-state Raman spectra can be obtained within seconds of signal averaging while being entirely insensitive to fluorescence This means that BV binding process, ranging from minutes to tens of minutes[23], can be followed in real time after mixing apoprotein with BV.
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