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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.


(A) Absorbancespectra for RpBphP2 PAS-GAF-PHY, PAS-GAF, PAS-GAF-PHYD202A, and PAS-GAF-PHY P465T mutants in the Pr state (black line)and photoconverted state (gray line); (B) light-minus-dark differenceabsorption spectra of RpBphP2 wild-type PAS-GAF-PHY (blue line), PAS-GAF(red line), D202A (magenta), and P465T (green line).
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fig3: (A) Absorbancespectra for RpBphP2 PAS-GAF-PHY, PAS-GAF, PAS-GAF-PHYD202A, and PAS-GAF-PHY P465T mutants in the Pr state (black line)and photoconverted state (gray line); (B) light-minus-dark differenceabsorption spectra of RpBphP2 wild-type PAS-GAF-PHY (blue line), PAS-GAF(red line), D202A (magenta), and P465T (green line).

Mentions: To further explore the origin of the prominent 1630 cm–1 (−)/1653 (+) cm–1 signal, we performedFTIR spectroscopy on a shorter construct, RpBphP2 PAS-GAF (also referredto as the chromophore binding domain (CBD)), and on the RpBphP2 PAS-GAF-PHYD202A mutant. Both proteins are deficient in Pfr formation. Upon photoconversion,the D202A mutant becomes arrested in a Meta-R-like state in whichthe absorption characteristic of the Pr state is bleached and onlya small induced absorption is evident, less red-shifted than thatin the Pfr state of wild-type11,13 (Figure 3). The PAS-GAF protein converts to a red-shifted state thatabsorbs at 741 nm11 and fails to form thefully red-shifted Pfr state of wild-type PAS-GAF-PHY, which absorbsat 753 nm (Figure 3).11 The FTIR difference spectra of the PAS-GAF (Figure 2, red line) and the D202A mutant (Figure 2, blue line) proteins are very similar. Strikingly, they bothmostly lack the negative band at 1630 cm–1 and thepositive band at 1653 cm–1 characteristic of wild-typePAS-GAF-PHY. Evidently, the β-sheet-to-α-helix switchdoes not occur in these proteins. Given that Asp-202 links the BVchromophore to the PHY domain14 and hasproved central in the protonation–deprotonation cycle of BV,13,28 this experimental result strongly suggests that the β-sheet-to-α-helixswitch observed in wild-type PAS-GAF-PHY takes place in the PHY domain.


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)

(A) Absorbancespectra for RpBphP2 PAS-GAF-PHY, PAS-GAF, PAS-GAF-PHYD202A, and PAS-GAF-PHY P465T mutants in the Pr state (black line)and photoconverted state (gray line); (B) light-minus-dark differenceabsorption spectra of RpBphP2 wild-type PAS-GAF-PHY (blue line), PAS-GAF(red line), D202A (magenta), and P465T (green line).
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fig3: (A) Absorbancespectra for RpBphP2 PAS-GAF-PHY, PAS-GAF, PAS-GAF-PHYD202A, and PAS-GAF-PHY P465T mutants in the Pr state (black line)and photoconverted state (gray line); (B) light-minus-dark differenceabsorption spectra of RpBphP2 wild-type PAS-GAF-PHY (blue line), PAS-GAF(red line), D202A (magenta), and P465T (green line).
Mentions: To further explore the origin of the prominent 1630 cm–1 (−)/1653 (+) cm–1 signal, we performedFTIR spectroscopy on a shorter construct, RpBphP2 PAS-GAF (also referredto as the chromophore binding domain (CBD)), and on the RpBphP2 PAS-GAF-PHYD202A mutant. Both proteins are deficient in Pfr formation. Upon photoconversion,the D202A mutant becomes arrested in a Meta-R-like state in whichthe absorption characteristic of the Pr state is bleached and onlya small induced absorption is evident, less red-shifted than thatin the Pfr state of wild-type11,13 (Figure 3). The PAS-GAF protein converts to a red-shifted state thatabsorbs at 741 nm11 and fails to form thefully red-shifted Pfr state of wild-type PAS-GAF-PHY, which absorbsat 753 nm (Figure 3).11 The FTIR difference spectra of the PAS-GAF (Figure 2, red line) and the D202A mutant (Figure 2, blue line) proteins are very similar. Strikingly, they bothmostly lack the negative band at 1630 cm–1 and thepositive band at 1653 cm–1 characteristic of wild-typePAS-GAF-PHY. Evidently, the β-sheet-to-α-helix switchdoes not occur in these proteins. Given that Asp-202 links the BVchromophore to the PHY domain14 and hasproved central in the protonation–deprotonation cycle of BV,13,28 this experimental result strongly suggests that the β-sheet-to-α-helixswitch observed in wild-type PAS-GAF-PHY takes place in the PHY domain.

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.