An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway |
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| Authors: | Sean R Tachibana Longteng Tang Liangdong Zhu Yuka Takeda Keiji Fushimi Yoshibumi Ueda Takahiro Nakajima Yuto Kuwasaki Moritoshi Sato Rei Narikawa Chong Fang |
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| Affiliation: | 1.Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331-4003, USA; (S.R.T.); (L.T.); (L.Z.);2.Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; (Y.T.); (K.F.); (R.N.);3.Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan; (Y.U.); (T.N.); (Y.K.); (M.S.) |
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| Abstract: | Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm−1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances. |
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| Keywords: | far-red/orange cyanobacteriochromes structure-activity relationships time-resolved spectroscopy reversible photoswitching optogenetics |
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