Vibrational Assignment of the Ultrafast Infrared Spectrum of the Photoactivatable Flavoprotein AppA

Haigney, Allison; Lukács, András; Brust, Richard; Zhao, Rui-Kun; Towrie, Michael; Greetham, Gregory M.; Clark, Ian P.; Illarionov, Boris; Bacher, Adelbert; Kim, Ryu-Ryun; Fischer, Markus; Meech, Stephen R.; Tonge, Peter J.

doi:10.1021/jp305220m

Cited by 23 publications

(65 citation statements)

References 31 publications

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“…9,11,18 In addition, the bleach observed at 1670 cm –1 in lBlsA is absent in both dF32N and lF32N BlsA, while a strong bleach is observed at 1642(3) cm –1 in both dF32N BlsA and lF32N BlsA. The 1642(3) cm –1 band is similar to the frequency of the C2=O observed in AppA BLUF 11,12 and is unaffected by 13 C labeling of the protein, indicating that it is a flavin mode. Because isotopic editing of the flavin chromophore revealed that the 1670 cm –1 bleach in wild-type lBlsA is the C2=O carbonyl, the simplest explanation is that the Asn at position 32 in F32N BlsA H-bonds to the C2=O, resulting in a red shift in the frequency of this vibration.…”

supporting

confidence: 62%

“…9,10 Because BlsA is a photoactive BLUF protein and also has the conserved glutamine (Q51, Figure S3, Supporting Information), this transient mode must either be absent or obscured in dBlsA. Similar to AppA BLUF , 9,11,12 a bleach at 1700 cm –1 in dBlsA is shifted by 10 cm –1 to 1690 cm –1 in lBlsA and is assigned as the C4=O carbonyl of the flavin. A decrease in frequency is indicative of an increase of H-bonding to the flavin in the light-adapted state.…”

mentioning

confidence: 99%

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Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Brust

Haigney

Lukács

et al. 2013

J. Phys. Chem. Lett.

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Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the β5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling.

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supporting

confidence: 62%

mentioning

confidence: 99%

Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Brust

Haigney

Lukács

et al. 2013

J. Phys. Chem. Lett.

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“…The next two bleaches at lower wavenumber (1630, 1585 cm -1 ) are ring modes with significant C4aN5 stretch contributions, and the most intense bleach (1550 cm -1 ) is a C10aN1 dominated mode. 34-36 The three transient absorptions (1385, 1430, 1615 cm -1 ) in the 2 ps spectrum are due to vibrations of the FMN singlet excited state, 1 FMN*. Significantly, two additional bleaches are well resolved in the 2 ps spectrum of AsLOV2 (at 1690 and 1670 cm -1 ), which must arise from vibrational modes of amino acids interacting with the chromophore.…”

Section: Resultsmentioning

confidence: 96%

“…2A shows marked differences between TRIR spectra of aqueous FMN and AsLOV2, with the latter revealing much richer structure in the 1600 – 1750 cm -1 region. The vibrational spectrum of FMN itself has been assigned; 34-36 the bleaches at 1660 cm -1 and 1700 cm -1 (Fig. 2A) are associated with C=O vibrations of isoalloxazine, which comprise a coupled pair with the higher wavenumber mode more localized on C4=O and the lower on C2=O.…”

Section: Resultsmentioning

confidence: 99%

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain

et al. 2017

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The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatio-temporal control of cell signalling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds timescale, which then modulate the activity of output domains responsible for biological signalling. Using time resolved vibrational spectroscopy coupled with isotope labelling we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 femtoseconds and one millisecond after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labelled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a sub-picosecond perturbation of the protein matrix occurs. In this perturbed environment the previously characterised reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the β-sheet then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signalling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

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“…These data are compared with TRIR of AppA BLUF mutants and model flavins which unambiguously display the spectra of radical intermediates. 55−57 In this way marker bands for neutral and radical states are identified. These assignments are confirmed by isotope substitution.…”

Section: Introductionmentioning

confidence: 99%

BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

et al. 2014

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BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered.

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Vibrational Assignment of the Ultrafast Infrared Spectrum of the Photoactivatable Flavoprotein AppA

Cited by 23 publications

References 31 publications

Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain

BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

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