QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors

Andrikopoulos, Prokopis C.; Chaudhari, Aditya S.; Liu, Yingliang; Konold, Patrick E.; Kennis, John T. M.; Schneider, Bohdan; Fuertes, Gustavo

doi:10.1039/d1cp00447f

Cited by 7 publications

(21 citation statements)

References 97 publications

(162 reference statements)

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“…The two carbonyl stretching modes of flavins (C2=O2 and C4=O2, see Fig. S2A ) also show up in the 1750 to 1650 cm -1 spectral region ( 8, 37, 38 ). Absolute FTIR spectra in the 1750-1500 cm -1 region of dark-adapted and lit-adapted wt EL222 in H 2 O are hardly distinguishable ( Fig.…”

Section: Resultsmentioning

confidence: 96%

“…In contrast, lit-minus-dark difference spectra show significant changes between both ( Figure 1B bottom right). The positive/negative features at 1727 cm -1 (+)/1713 cm -1 (-) and 1684 cm -1 (+)/1700 cm -1 (-) can be attributed to the C4=O4 and C2=O2 stretches of FMN, respectively ( 8, 39, 40 ). The positive-negative-positive feature at 1551 cm -1 (+)/1534 cm -1 (-)/1516 cm -1 (+) is probably due to changes in the protein amide II band, but potential contributions from side-chains (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…In the dark-to-lit transition of EL222 measured in D2O, the time-resolved IR data is typically analyzed using a sequential kinetic model according to which a singlet species ( 1 FMN*) is nearly instantaneously formed upon photon absorption, followed by the triplet species in ∼3 ns ( 3 FMN*), and finally the adduct state in ∼5 μs (A390) ( 5, 8 ). Because the steady-state difference spectra are different from the transient spectra of the adduct state obtained a few microseconds after irradiation ( Figure S31 ), the existence of an additional intermediate species in the multi-millisecond time scale is envisioned, as previously postulated ( 5 ).…”

Section: Resultsmentioning

confidence: 99%

“…The nearby amide II band (1600 to 1500 cm -1 ) is due to C-N stretching and N-H bending modes, which can be used to report on solvent accessibility of the protein backbone via hydrogen/deuterium exchange experiments (36). The two carbonyl stretching modes of flavins (C2=O2 and C4=O2, see Figure S2A) also show up in the 1750 to 1650 cm -1 spectral region (8,37,38). Absolute FTIR spectra in the 1750-1500 cm -1 region of dark-adapted and lit-adapted wt EL222 in H2O are hardly distinguishable (Figure 1B top right).…”

Section: Ir Absorption Region Of Native Protein and Fmnmentioning

confidence: 99%

“…This frequency shift is caused by photon absorption by the oxidized FMN cofactor, which triggers its partial reduction to a semiquinone radical and adduct formation with a conserved cysteine residue ( 7 ). FMN-Cys covalent bond formation takes place in the nanosecond to microsecond time scale ( 5, 8 ). The rupture of such an adduct state, sometimes referred to as A 390 species ( 5 ), is much slower, typically returning to the resting state in seconds to hours, depending on the particular LOV domain ( 9 ).…”

Section: Introductionmentioning

confidence: 99%

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Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Chaudhari

Chatterjee

Domingos

et al. 2022

Preprint

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Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at forty-five positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/Visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A’α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A’α extension of the LOV domain in controlling EL222 photocycle length.SIGNIFICANCEThe kinetics of fold switching between non-illuminated and blue-light-irradiated states in the transcription factor EL222 is important for understanding the signal transduction mechanism of LOV photoreceptors. Here we combine two native probes, the FMN chromophore (absorption bands in the UV/Visible region) and the protein backbone (amide bands in the infrared region), with genetically encoded cyano (C≡ N)-containing phenylalanine residues as infrared reporters of protein microenvironments. EL222 structural dynamics is more complex than expected if using a single type of probe. Local changes around residues 31 and 35 precede FMN-protein adduct rupture, which in turn precedes the global protein conformational relaxation. Our findings point the way forward to obtaining comprehensive descriptions of kinetic transitions in LOV and other photosensors.TEASERFold switching kinetics from lit to dark state in a photoreceptor is revealed by infrared-active non-canonical amino acids.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Ir Absorption Region Of Native Protein and Fmnmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Chaudhari

Chatterjee

Domingos

et al. 2022

Preprint

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Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields

et al. 2023

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We present time‐resolved Gd−Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter‐residue distances during a protein's mechanical cycle in the solution state. TiGGER makes use of Gd‐sTPATCN spin labels, whose favorable qualities include a spin‐7/2 EPR‐active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C‐terminus and N‐terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light‐activated long‐range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

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Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields

et al. 2023

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QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors

Abstract: The isomerisation of a conserved glutamine residue along the early photocycle of 3 LOV domains is explored by QM calculations, revealing IR spectral shifts upon rotation in agreement with time-resolved experiments.

Cited by 7 publications

References 97 publications

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields

Triggered Functional Dynamics of AsLOV2 by Time‐Resolved Electron Paramagnetic Resonance at High Magnetic Fields

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