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Elucidating Ultrafast Multiphasic Dynamics in the Photoisomerization of Cyanobacteriochrome.

ABSTRACT: Understanding photoisomerization dynamics in cyanobacteriochromes is important to the development of optical agents in near-infrared biological imaging and optogenetics. Here, by integrating femtosecond spectroscopy and site-directed mutagenesis, we investigate the photoinduced Pr-state isomerization dynamics and mechanism of a unique red/green cyanobacteriochrome from Leptolyngbya sp. JSC-1. We observed multiphasic dynamics in the Pr state, a widespread phenomenon for photoreceptors in the phytochrome superfamily, and revealed their origins; the initial dynamics over a few to tens and hundreds of picoseconds arises from the local active-site relaxations followed by the slow double-bond isomerization in several hundreds of picoseconds. Such continuous active-site evolution results in a unique spectral tuning effect that favors the blue-side emission and suppresses the red-side emission. We also observed the faster dynamics in both relaxation and isomerization with critical mutants at the active site that render a looser active site. These results clearly distinguish the multiphasic dynamics between relaxation and isomerization and reveal a novel molecular mechanism for better biological applications.

SUBMITTER: Wang D

PROVIDER: S-EPMC8172094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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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Elucidating Ultrafast Multiphasic Dynamics in the Photoisomerization of Cyanobacteriochrome.

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