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How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

Nohr D, Rodriguez R, Weber S, Schleicher E - Front Mol Biosci (2015)

Bottom Line: An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given.In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains.Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg Freiburg, Germany.

ABSTRACT
Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

No MeSH data available.


TrEPR spectra of various BLUF domains recorded at 1 μs after pulsed laser excitation (adapted from Weber et al., 2011). Upper: dark-adapted and blue-light adapted AppA-BLUF protein. Middle: dark and blue-light illuminated YcgF-BLUF samples. Lower: dark and blue-light illuminated Slr-BLUF protein. The respective dashed curves represent spectral simulations of the dark-state sample.
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Figure 5: TrEPR spectra of various BLUF domains recorded at 1 μs after pulsed laser excitation (adapted from Weber et al., 2011). Upper: dark-adapted and blue-light adapted AppA-BLUF protein. Middle: dark and blue-light illuminated YcgF-BLUF samples. Lower: dark and blue-light illuminated Slr-BLUF protein. The respective dashed curves represent spectral simulations of the dark-state sample.

Mentions: Photo-induced flavin triplet states and RP species have been detected on a microsecond time scale, but revealed a completely different behavior as compared to previously investigated LOV domains (Schleicher et al., 2004). Moreover, BLUF domains exhibit much higher spectral diversity. While the trEPR spectrum of the AppA-BLUF protein (see Figure 5, upper panel) is very similar to that of the LOV domains, triplet-state spectra obtained from the YgcF-BLUF protein show a completely different electron-spin polarization pattern (see Figure 5, middle panel). Moreover, spectra recorded from the Slr-BLUF protein exhibit an even more complex spectral shape around g ~ 2 (see Figure 5, lower panel). To rationalize these differences, spectral simulations of flavin-triplet state trEPR spectra have been performed (see dashed lines in Figure 5) (Weber et al., 2011). It is interesting to note that pre-illuminated BLUF domains did not show any signal assuming an efficient deactivation process that is completed within a ns-time scale (Mathes et al., 2012).


How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

Nohr D, Rodriguez R, Weber S, Schleicher E - Front Mol Biosci (2015)

TrEPR spectra of various BLUF domains recorded at 1 μs after pulsed laser excitation (adapted from Weber et al., 2011). Upper: dark-adapted and blue-light adapted AppA-BLUF protein. Middle: dark and blue-light illuminated YcgF-BLUF samples. Lower: dark and blue-light illuminated Slr-BLUF protein. The respective dashed curves represent spectral simulations of the dark-state sample.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4555020&req=5

Figure 5: TrEPR spectra of various BLUF domains recorded at 1 μs after pulsed laser excitation (adapted from Weber et al., 2011). Upper: dark-adapted and blue-light adapted AppA-BLUF protein. Middle: dark and blue-light illuminated YcgF-BLUF samples. Lower: dark and blue-light illuminated Slr-BLUF protein. The respective dashed curves represent spectral simulations of the dark-state sample.
Mentions: Photo-induced flavin triplet states and RP species have been detected on a microsecond time scale, but revealed a completely different behavior as compared to previously investigated LOV domains (Schleicher et al., 2004). Moreover, BLUF domains exhibit much higher spectral diversity. While the trEPR spectrum of the AppA-BLUF protein (see Figure 5, upper panel) is very similar to that of the LOV domains, triplet-state spectra obtained from the YgcF-BLUF protein show a completely different electron-spin polarization pattern (see Figure 5, middle panel). Moreover, spectra recorded from the Slr-BLUF protein exhibit an even more complex spectral shape around g ~ 2 (see Figure 5, lower panel). To rationalize these differences, spectral simulations of flavin-triplet state trEPR spectra have been performed (see dashed lines in Figure 5) (Weber et al., 2011). It is interesting to note that pre-illuminated BLUF domains did not show any signal assuming an efficient deactivation process that is completed within a ns-time scale (Mathes et al., 2012).

Bottom Line: An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given.In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains.Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg Freiburg, Germany.

ABSTRACT
Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

No MeSH data available.