Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome

Kübel, Joachim; Chenchiliyan, Manoop; Ooi, Saik Ann; Gustavsson, Emil; Isaksson, Linnéa; Kuznetsova, Valentyna; Ihalainen, Janne A.; Westenhoff, Sebastian; Maj, Michał

doi:10.1039/c9cp06995j

Cited by 24 publications

(41 citation statements)

References 53 publications

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“…Such a CI is reached through a counterclockwise rotation of the D-ring with a hula-twist mechanism, resulting in a structure that is here interpreted as the early Lumi-R intermediate. Notably, this intermediate is characterized by the interaction of the chromophore D-ring with a tyrosine residue (Tyr263) in line with mutational studies 38,46 that demonstrated the tight connection between Tyr263 and the chromophore state. The IR spectrum calculated on the proposed structure shows a good agreement with the experimental data.…”

Section: Introductionmentioning

confidence: 53%

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Photochemistry and transient intermediates in a bacteriophytochrome photocycle revealed by multiscale simulations

Salvadori

Macaluso

Pellicci

et al. 2022

Preprint

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Phytochromes are photoreceptors responsible for sensing light in plants, fungi and bacteria. Their photoactivation is initiated by the photoisomerization of an embedded chromophore, which triggers a large conformational change in the structure of the entire protein. Although phytochromes have been subject of numerous studies, the photoisomerization mechanism and the following reaction path leading to the final active state remain elusive. Here, we use an integrated computational approach that combines non-adiabatic surface hopping and adiabatic ground-state molecular dynamics simulations to gain atomistic details on the photoactivation mechanism of Deinococcus radiodurans bacteriophytochrome. Our simulations show that the ps-scale photoisomerization of the chromophore proceeds through a hula-twist mechanism that forces a counterclockwise rotation of the D-ring. The initial photoproduct rapidly evolves in an early intermediate which we characterize through IR spectroscopy simulation. The early intermediate then evolves on the nanosecond-to-microsecond scale to a late intermediate, characterized by a more disordered binding pocket and a clear weakening of the aspartate-to-arginine salt bridge interaction, whose cleavage is essential to interconvert to the final active state.

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Section: Introductionmentioning

confidence: 53%

“…We assign this conformation to the early Lumi-R state. Mutagenesis studies 38,46 have suggested that the Y263F mutation hinders the formation of Lumi-R, thereby reducing the photoconversion (Pr→Pfr) yield: our simulations show that Tyr263 is indeed fundamental for stabilizing the Lumi-R state through a hydrogen bond with the N D atom of BV.…”

Section: 71mentioning

confidence: 63%

Photochemistry and transient intermediates in a bacteriophytochrome photocycle revealed by multiscale simulations

Salvadori

Macaluso

Pellicci

et al. 2022

Preprint

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“…Mutational studies have identified highly conserved key amino acids (e.g., Y133, Y47, D79 and H160 in Syn Cph2 or Y263, Y176, D207 and H290 in Syn Cph1 and DrBphP) in the immediate vicinity of the D-ring that directly affect the excited state lifetime and the isomerization quantum yield [ 10 , 13 , 36 , 42 , 60 , 61 , 62 , 63 ]. In case of the Tyr residues both their steric interactions with the chromophore as well as their participation in the hydrogen bonding network have been proposed as essential for efficient photochemistry [ 60 , 62 , 63 , 64 , 65 ]. Additionally, solvent exposure, the overall hydrogen bonding network and the protonation state of several residues inside the pocket also appear to be important for its properties [ 34 , 36 , 42 , 43 , 60 , 61 , 66 ] and the ensuing kinetics.…”

Section: Resultsmentioning

confidence: 99%

Ultrafast Photoconversion Dynamics of the Knotless Phytochrome SynCph2

Fischer

Wilderen

Gnau

et al. 2021

IJMS

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The family of phytochrome photoreceptors contains proteins with different domain architectures and spectral properties. Knotless phytochromes are one of the three main subgroups classified by their distinct lack of the PAS domain in their photosensory core module, which is in contrast to the canonical PAS-GAF-PHY array. Despite intensive research on the ultrafast photodynamics of phytochromes, little is known about the primary kinetics in knotless phytochromes. Here, we present the ultrafast Pr ⇆ Pfr photodynamics of SynCph2, the best-known knotless phytochrome. Our results show that the excited state lifetime of Pr* (~200 ps) is similar to bacteriophytochromes, but much longer than in most canonical phytochromes. We assign the slow Pr* kinetics to relaxation processes of the chromophore-binding pocket that controls the bilin chromophore’s isomerization step. The Pfr photoconversion dynamics starts with a faster excited state relaxation than in canonical phytochromes, but, despite the differences in the respective domain architectures, proceeds via similar ground state intermediate steps up to Meta-F. Based on our observations, we propose that the kinetic features and overall dynamics of the ultrafast photoreaction are determined to a great extent by the geometrical context (i.e., available space and flexibility) within the binding pocket, while the general reaction steps following the photoexcitation are most likely conserved among the red/far-red phytochromes.

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“…The understanding of the phytochrome family has been largely driven by experimental studies employing IR, 20 resonance Raman, 21 − 24 NMR, 25 and X-ray crystallography. 17 , 26 − 29 In contrast to other photoactive systems such as rhodopsin and photoactive yellow protein, there are few recent computational studies attempting to determine how the signal is relayed from the chromophore to the protein matrix.…”

Section: Introductionmentioning

confidence: 99%

“…However, this goal poses a major difficulty due to the so-called the Boltzmann sampling problem 39 caused mainly due to the existence of long time scales involved in the dynamics of photoproducts. 20 To achieve such a challenging goal, we employ enhanced sampling MD simulations to overcome the FE barrier of the primary photoisomerization event and to efficiently sample nonequilibrium configurations visited by the BphP-BV complex in the Pr and Pfr conformers. By performing simulations with an aggregate time of ∼2 μs, we study the Pr and Pfr conformers starting from their equilibrium configurations, i.e., before the photoisomerization takes place (Pr) and after the primary photochemical event is concluded (Pfr).…”

Section: Introductionmentioning

confidence: 99%

Enhancing the Inhomogeneous Photodynamics of Canonical Bacteriophytochrome

et al. 2022

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The ability of phytochromes to act as photoswitches in plants and microorganisms depends on interactions between a bilin-like chromophore and a host protein. The interconversion occurs between the spectrally distinct red (Pr) and far-red (Pfr) conformers. This conformational change is triggered by the photoisomerization of the chromophore D-ring pyrrole. In this study, as a representative example of a phytochrome-bilin system, we consider biliverdin IXα (BV) bound to bacteriophytochrome (BphP) from Deinococcus radiodurans . In the absence of light, we use an enhanced sampling molecular dynamics (MD) method to overcome the photoisomerization energy barrier. We find that the calculated free energy (FE) barriers between essential metastable states agree with spectroscopic results. We show that the enhanced dynamics of the BV chromophore in BphP contributes to triggering nanometer-scale conformational movements that propagate by two experimentally determined signal transduction pathways. Most importantly, we describe how the metastable states enable a thermal transition known as the dark reversion between Pfr and Pr, through a previously unknown intermediate state of Pfr. We present the heterogeneity of temperature-dependent Pfr states at the atomistic level. This work paves a way toward understanding the complete mechanism of the photoisomerization of a bilin-like chromophore in phytochromes.

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Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome

Abstract: Infra-red spectroscopy advances our understanding of how photosensory proteins carry their function.

Cited by 24 publications

References 53 publications

Photochemistry and transient intermediates in a bacteriophytochrome photocycle revealed by multiscale simulations

Photochemistry and transient intermediates in a bacteriophytochrome photocycle revealed by multiscale simulations

Ultrafast Photoconversion Dynamics of the Knotless Phytochrome SynCph2

Enhancing the Inhomogeneous Photodynamics of Canonical Bacteriophytochrome

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