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Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

ABSTRACT: Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state ( ^15Z Pb) and orange-absorbing 15E state ( ^15E Po). pH titration analysis revealed that ^15Z Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, ^15E Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state ( ^15Z Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in ^15Z Pb and bilin protonation in ^15E Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated ^15Z Pg and protonated ^15E Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed ^15Z Pg formation as an intermediate in the ^15E Pr-to- ^15Z Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies.

SUBMITTER: Sato T

PROVIDER: S-EPMC6901293 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

Sato Teppei T Kikukawa Takashi T Miyoshi Risako R Kajimoto Kousuke K Yonekawa Chinatsu C Fujisawa Tomotsumi T Unno Masashi M Eki Toshihiko T Hirose Yuu Y

The Journal of biological chemistry 20191024 49

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo <i>15Z/15E</i> bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely ...[more]

PMID: 31649035

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Project description:SignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.AimWe describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.ApproachWe used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices.ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.ConclusionT-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

Dataset Information

Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

Publications

Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

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