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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.


Pulsed X-Band proton Davies-type ENDOR spectra of various AsLOV2 single- and double mutants (adapted from Brosi et al., 2010). Left: Spectra were recorded at 120 K (dashed lines), 80 K (black lines), and 10 K (gray shaded) for all samples. Right: Sections of AsLOV2 10-K spectra with accompanying spectral simulation of the outer wing of the spectrum (measured spectrum, dashed lines; single simulated hfcs, shades of blue, green, and red; envelope of simulated hfcs, black lines). Two protein samples, AsLOV2 C450A/F509A and AsLOV2 C450A/N425C, require another hyperfine component of axial symmetry for accurate spectral fitting. This feature represents hfcs from fast rotating 8α methyl group protons, is shown in purple and is denoted as H8αiso.
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Figure 4: Pulsed X-Band proton Davies-type ENDOR spectra of various AsLOV2 single- and double mutants (adapted from Brosi et al., 2010). Left: Spectra were recorded at 120 K (dashed lines), 80 K (black lines), and 10 K (gray shaded) for all samples. Right: Sections of AsLOV2 10-K spectra with accompanying spectral simulation of the outer wing of the spectrum (measured spectrum, dashed lines; single simulated hfcs, shades of blue, green, and red; envelope of simulated hfcs, black lines). Two protein samples, AsLOV2 C450A/F509A and AsLOV2 C450A/N425C, require another hyperfine component of axial symmetry for accurate spectral fitting. This feature represents hfcs from fast rotating 8α methyl group protons, is shown in purple and is denoted as H8αiso.

Mentions: In detail, it was possible to detect a unique spectral behavior of Avena sativa LOV2 (AsLOV2) C450A samples as their 8α methyl-group rotational motion is slowed down starting at already rather elevated temperatures (T < 110 K) (Brosi et al., 2010). To identify responsible amino acids for altered protein-cofactor interaction, an extended mutagenesis study has been performed with modifications introduced to in the direct vicinity of the 8α methyl-group, where three amino acids, namely Leu496, Phe509, and Asn425, are located (see Figure 4). Mutations in these three amino acids clearly showed changed temperature behavior, which is in line with the predicted altered sterical interaction (see Figure 4, left panel). Moreover, the hfcs from the three arrested 8α protons shift as depending on the individual mutants (see Figure 4, right panel). Spectral assignment of these hfc tensors in combination with DFT calculations resulted in the precise determination of the orientation of the methyl group with respect to the isoalloxazine ring plane.


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)

Pulsed X-Band proton Davies-type ENDOR spectra of various AsLOV2 single- and double mutants (adapted from Brosi et al., 2010). Left: Spectra were recorded at 120 K (dashed lines), 80 K (black lines), and 10 K (gray shaded) for all samples. Right: Sections of AsLOV2 10-K spectra with accompanying spectral simulation of the outer wing of the spectrum (measured spectrum, dashed lines; single simulated hfcs, shades of blue, green, and red; envelope of simulated hfcs, black lines). Two protein samples, AsLOV2 C450A/F509A and AsLOV2 C450A/N425C, require another hyperfine component of axial symmetry for accurate spectral fitting. This feature represents hfcs from fast rotating 8α methyl group protons, is shown in purple and is denoted as H8αiso.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555020&req=5

Figure 4: Pulsed X-Band proton Davies-type ENDOR spectra of various AsLOV2 single- and double mutants (adapted from Brosi et al., 2010). Left: Spectra were recorded at 120 K (dashed lines), 80 K (black lines), and 10 K (gray shaded) for all samples. Right: Sections of AsLOV2 10-K spectra with accompanying spectral simulation of the outer wing of the spectrum (measured spectrum, dashed lines; single simulated hfcs, shades of blue, green, and red; envelope of simulated hfcs, black lines). Two protein samples, AsLOV2 C450A/F509A and AsLOV2 C450A/N425C, require another hyperfine component of axial symmetry for accurate spectral fitting. This feature represents hfcs from fast rotating 8α methyl group protons, is shown in purple and is denoted as H8αiso.
Mentions: In detail, it was possible to detect a unique spectral behavior of Avena sativa LOV2 (AsLOV2) C450A samples as their 8α methyl-group rotational motion is slowed down starting at already rather elevated temperatures (T < 110 K) (Brosi et al., 2010). To identify responsible amino acids for altered protein-cofactor interaction, an extended mutagenesis study has been performed with modifications introduced to in the direct vicinity of the 8α methyl-group, where three amino acids, namely Leu496, Phe509, and Asn425, are located (see Figure 4). Mutations in these three amino acids clearly showed changed temperature behavior, which is in line with the predicted altered sterical interaction (see Figure 4, left panel). Moreover, the hfcs from the three arrested 8α protons shift as depending on the individual mutants (see Figure 4, right panel). Spectral assignment of these hfc tensors in combination with DFT calculations resulted in the precise determination of the orientation of the methyl group with respect to the isoalloxazine ring plane.

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.