Limits...
FTIR Spectroscopy Revealing Light-Dependent Refolding of the Conserved Tongue Region of Bacteriophytochrome.

Stojković EA, Toh KC, Alexandre MT, Baclayon M, Moffat K, Kennis JT - J Phys Chem Lett (2014)

Bottom Line: Our results indicate conversion from a β-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (Takala H.; et al.Nature2014, 5, 245-248).A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the β-sheet-to-α-helix conversion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, The University of Chicago , Chicago, Illinois 60637, United States.

ABSTRACT
Bacteriophytochromes (BphPs) constitute a class of photosensory proteins that toggle between Pr and Pfr functional states through absorption of red and far-red light. The photosensory core of BphPs is composed of PAS, GAF, and PHY domains. Here, we apply FTIR spectroscopy to investigate changes in the secondary structure of Rhodopseudomonas palustris BphP2 (RpBphP2) upon Pr to Pfr photoconversion. Our results indicate conversion from a β-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (Takala H.; et al. Nature2014, 5, 245-248). A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the β-sheet-to-α-helix conversion.

No MeSH data available.


Light-minus-dark FTIR spectra of RpBphP2PAS-GAF-PHY (black), PAS-GAF(red), PAS-GAF-PHY D202A mutant (blue), and PAS-GAF-PHY P465T mutant(green).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126705&req=5

fig2: Light-minus-dark FTIR spectra of RpBphP2PAS-GAF-PHY (black), PAS-GAF(red), PAS-GAF-PHY D202A mutant (blue), and PAS-GAF-PHY P465T mutant(green).

Mentions: Sample preparation, site-directed mutagenesis,and FTIR spectroscopyon BphPs were carried out as described before.11,17 Figure 2 shows the light-minus-dark FTIRspectra of the RpBphP2 PAS-GAF-PHY photosensory core domain (black),the PAS-GAF chromophore-binding domain (red), the PAS-GAF-PHY D202Amutant (blue), and the PAS-GAF-PHY P465T mutant (green). D202 in RpBphP2represents a well-characterized, conserved Asp from the PASDIP motifof the GAF domain, while P465 represents a conserved Pro from thePRXSF motif of the PHY domain. The spectra were scaled on the highest-wavenumberC1=O bleach near 1738 cm–1. For PAS-GAF-PHY,the spectrum represents the Pfr–Pr spectrum. The FTIR differencespectrum shows a multitude of positive and negative bands, some ofwhich were assigned before in this protein, RpBphP2.17,18 BV-specific bands were identified before on the basis of ultrafastIR experiments on RpBphP2, where optical excitation results in BV-specificsignals on femtosecond and picosecond time scales before any signalsfrom the apoprotein arise.17,19,20 Other BV-specific bands were observed and assigned before in Cph1on the basis of isotope labeling of the chromophore and apoprotein21 and will be discussed below. Here, we focusattention on the highest-amplitude features, the negative band at1630 cm–1 and positive band at 1653 cm–1. Similar bands were also observed in Cph1 and assigned to amideI.21 Given the amide I wavenumber rangeswhere distinct secondary structure elements have their main absorption,22 this signal corresponds to a loss of a β-sheet(negative signal at 1630 cm–1) and a gain of anα-helix (positive signal at 1653 cm–1) upontransformation of Pr into Pfr.


FTIR Spectroscopy Revealing Light-Dependent Refolding of the Conserved Tongue Region of Bacteriophytochrome.

Stojković EA, Toh KC, Alexandre MT, Baclayon M, Moffat K, Kennis JT - J Phys Chem Lett (2014)

Light-minus-dark FTIR spectra of RpBphP2PAS-GAF-PHY (black), PAS-GAF(red), PAS-GAF-PHY D202A mutant (blue), and PAS-GAF-PHY P465T mutant(green).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126705&req=5

fig2: Light-minus-dark FTIR spectra of RpBphP2PAS-GAF-PHY (black), PAS-GAF(red), PAS-GAF-PHY D202A mutant (blue), and PAS-GAF-PHY P465T mutant(green).
Mentions: Sample preparation, site-directed mutagenesis,and FTIR spectroscopyon BphPs were carried out as described before.11,17 Figure 2 shows the light-minus-dark FTIRspectra of the RpBphP2 PAS-GAF-PHY photosensory core domain (black),the PAS-GAF chromophore-binding domain (red), the PAS-GAF-PHY D202Amutant (blue), and the PAS-GAF-PHY P465T mutant (green). D202 in RpBphP2represents a well-characterized, conserved Asp from the PASDIP motifof the GAF domain, while P465 represents a conserved Pro from thePRXSF motif of the PHY domain. The spectra were scaled on the highest-wavenumberC1=O bleach near 1738 cm–1. For PAS-GAF-PHY,the spectrum represents the Pfr–Pr spectrum. The FTIR differencespectrum shows a multitude of positive and negative bands, some ofwhich were assigned before in this protein, RpBphP2.17,18 BV-specific bands were identified before on the basis of ultrafastIR experiments on RpBphP2, where optical excitation results in BV-specificsignals on femtosecond and picosecond time scales before any signalsfrom the apoprotein arise.17,19,20 Other BV-specific bands were observed and assigned before in Cph1on the basis of isotope labeling of the chromophore and apoprotein21 and will be discussed below. Here, we focusattention on the highest-amplitude features, the negative band at1630 cm–1 and positive band at 1653 cm–1. Similar bands were also observed in Cph1 and assigned to amideI.21 Given the amide I wavenumber rangeswhere distinct secondary structure elements have their main absorption,22 this signal corresponds to a loss of a β-sheet(negative signal at 1630 cm–1) and a gain of anα-helix (positive signal at 1653 cm–1) upontransformation of Pr into Pfr.

Bottom Line: Our results indicate conversion from a β-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (Takala H.; et al.Nature2014, 5, 245-248).A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the β-sheet-to-α-helix conversion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, The University of Chicago , Chicago, Illinois 60637, United States.

ABSTRACT
Bacteriophytochromes (BphPs) constitute a class of photosensory proteins that toggle between Pr and Pfr functional states through absorption of red and far-red light. The photosensory core of BphPs is composed of PAS, GAF, and PHY domains. Here, we apply FTIR spectroscopy to investigate changes in the secondary structure of Rhodopseudomonas palustris BphP2 (RpBphP2) upon Pr to Pfr photoconversion. Our results indicate conversion from a β-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (Takala H.; et al. Nature2014, 5, 245-248). A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the β-sheet-to-α-helix conversion.

No MeSH data available.