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Conventional in situ cryotrapping of phytochrome (PHY) intermediates has proven experimentally challenging for solid-state nuclear magnetic resonance (NMR) studies. To overcome this hindrance, we introduced the preparation of dehydrated disaccharide–PHY glasses, which allow transient photocycle intermediates to be isolated for NMR measurement at room temperature (RT). Here, we provide details of the incorporation of PHYs into a glassy matrix formed by trehalose. The protocol has been in routine use in our solid-state NMR studies for diverse PHYs having different molecular weights, bilin chromophore structures, and lifetimes of photoproducts. More recently, this protocol has been optimized for the isolation of individual transient reaction intermediates of a cyanobacteriochrome (CBCR) photosensor. We also provide information on Nuclear Magnetic Resonance(NMR) analysis to gain structural and dynamic information of the trehalose-embedded PHY proteins.

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Encapsulation of Phytochrome Proteins in Trehalose Matrices: Practical Considerations for the Use in Solid-State NMR Spectroscopy

  • 实验方案

  • 发表日期：

    2026年

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  • Valentin Rohr,
  • Wolfgang Gärtner,
  • Jörg Matysik,
  • Chen Song

摘要

Conventional in situ cryotrapping of phytochrome (PHY) intermediates has proven experimentally challenging for solid-state nuclear magnetic resonance (NMR) studies. To overcome this hindrance, we introduced the preparation of dehydrated disaccharide–PHY glasses, which allow transient photocycle intermediates to be isolated for NMR measurement at room temperature (RT). Here, we provide details of the incorporation of PHYs into a glassy matrix formed by trehalose. The protocol has been in routine use in our solid-state NMR studies for diverse PHYs having different molecular weights, bilin chromophore structures, and lifetimes of photoproducts. More recently, this protocol has been optimized for the isolation of individual transient reaction intermediates of a cyanobacteriochrome (CBCR) photosensor. We also provide information on Nuclear Magnetic Resonance(NMR) analysis to gain structural and dynamic information of the trehalose-embedded PHY proteins.