Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

Yusaku Hontani, Mikhail Baloban, Francisco Velazquez Escobar, Swetta A. Jansen, Daria M. Shcherbakova, Jörn Weißenborn, Miroslav Kloz, Maria Andrea Mroginski, Vladislav V. Verkhusha, John T.M. Kennis

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


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.

Original languageEnglish (US)
Article number3
JournalCommunications Chemistry
Issue number1
StatePublished - Dec 2021

ASJC Scopus subject areas

  • General Chemistry
  • Materials Chemistry
  • Biochemistry
  • Environmental Chemistry


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