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The photoinitiated reaction pathway of full-length cyanobacteriochrome Tlr0924 monitored over 12 orders of magnitude.

Hauck AF, Hardman SJ, Kutta RJ, Greetham GM, Heyes DJ, Scrutton NS - J. Biol. Chem. (2014)

Bottom Line: The CBCR Tlr0924 contains both phycocyanobilin and phycoviolobilin chromophores, and these two species photoisomerize in parallel via spectrally and kinetically equivalent intermediates before the second step of the photoreaction where the reaction pathways diverge, the loss of a thioether linkage to a conserved cysteine residue occurs, and the phycocyanobilin reaction terminates in a red-absorbing state, whereas the phycoviolobilin reaction proceeds more rapidly to a final green-absorbing state.Our analysis shows that Tlr0924 undergoes an unprecedented long photoreaction that spans from picoseconds to seconds.We show that the thermally driven, long timescale changes are less complex than those reported for the red/far-red photocycles of the related phytochrome photoreceptors.

View Article: PubMed Central - PubMed

Affiliation: From the Manchester Institute of Biotechnology and Photon Science Institute, Faculty of Life Sciences, The University of Manchester, Manchester M13 9PL, United Kingdom and.

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A, LED flash photolysis spectra at selected time points for PCB-only Tlr0924 with overlapping PVB forward and reverse reactions subtracted. B, global analysis of the data showing resulting EADS (black dots) fitted with a sum of Gaussian functions (red line). There are obvious features originating from the 15Z-PCB′Pb state (blue lines), the 15E-PCB′Pb state (cyan lines), the 15E-PCBPr state (dark red lines), and the 15E-PVBPg state. There is an additional feature at 640 nm (purple line) originating from inactive or modified protein. EADS1 converts to EADS2 with a lifetime of 3.1 ± 0.1 s.
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Figure 7: A, LED flash photolysis spectra at selected time points for PCB-only Tlr0924 with overlapping PVB forward and reverse reactions subtracted. B, global analysis of the data showing resulting EADS (black dots) fitted with a sum of Gaussian functions (red line). There are obvious features originating from the 15Z-PCB′Pb state (blue lines), the 15E-PCB′Pb state (cyan lines), the 15E-PCBPr state (dark red lines), and the 15E-PVBPg state. There is an additional feature at 640 nm (purple line) originating from inactive or modified protein. EADS1 converts to EADS2 with a lifetime of 3.1 ± 0.1 s.

Mentions: To investigate the properties of the PCB forward photoreaction in more detail, the pure PCB′ Pb state was regenerated by illuminating the 15E isomers (Pg and Pr) at 640 nm between each dataset collected. This converted only the PCB Pr population to the PCB′ Pb population whereas the PVB population remained in the Pg state. As with the mixture of PVB and PCB, after the initial photoisomerization observed in the ultrafast measurements there were no significant spectral changes between 20 ns and tens of ms (Fig. 5C). Due to the slow reaction dynamics and low concentrations of the “PCB” sample LED flash photolysis was used to monitor the dynamic processes. Samples were excited with a 10-ms, 455-nm LED flash in a UV-visible spectrophotometer to give the raw difference spectra shown in Fig. 6A. However, it is apparent that there were overlapping signals in the dataset, which originated from both the forward and reverse photoreactions of the PVB chromophore. Subtraction of scaled datasets collected on the same wavelength and time domains for the photoconversion of PVB from Pb to Pg (shown in Fig. 4A) and for the photoconversion of PVB from Pg to Pb (shown in Fig. 6B) resulted in the difference spectra shown in Fig. 7A, which should include components only from the PCB Pb to Pr photoconversion. Initial spectra show ground state bleaches at ∼325, 450, and 640 nm, and these spectra evolve over several seconds to a spectrum with a large bleach at ∼435 nm and a positive feature at ∼590 nm.


The photoinitiated reaction pathway of full-length cyanobacteriochrome Tlr0924 monitored over 12 orders of magnitude.

Hauck AF, Hardman SJ, Kutta RJ, Greetham GM, Heyes DJ, Scrutton NS - J. Biol. Chem. (2014)

A, LED flash photolysis spectra at selected time points for PCB-only Tlr0924 with overlapping PVB forward and reverse reactions subtracted. B, global analysis of the data showing resulting EADS (black dots) fitted with a sum of Gaussian functions (red line). There are obvious features originating from the 15Z-PCB′Pb state (blue lines), the 15E-PCB′Pb state (cyan lines), the 15E-PCBPr state (dark red lines), and the 15E-PVBPg state. There is an additional feature at 640 nm (purple line) originating from inactive or modified protein. EADS1 converts to EADS2 with a lifetime of 3.1 ± 0.1 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4067208&req=5

Figure 7: A, LED flash photolysis spectra at selected time points for PCB-only Tlr0924 with overlapping PVB forward and reverse reactions subtracted. B, global analysis of the data showing resulting EADS (black dots) fitted with a sum of Gaussian functions (red line). There are obvious features originating from the 15Z-PCB′Pb state (blue lines), the 15E-PCB′Pb state (cyan lines), the 15E-PCBPr state (dark red lines), and the 15E-PVBPg state. There is an additional feature at 640 nm (purple line) originating from inactive or modified protein. EADS1 converts to EADS2 with a lifetime of 3.1 ± 0.1 s.
Mentions: To investigate the properties of the PCB forward photoreaction in more detail, the pure PCB′ Pb state was regenerated by illuminating the 15E isomers (Pg and Pr) at 640 nm between each dataset collected. This converted only the PCB Pr population to the PCB′ Pb population whereas the PVB population remained in the Pg state. As with the mixture of PVB and PCB, after the initial photoisomerization observed in the ultrafast measurements there were no significant spectral changes between 20 ns and tens of ms (Fig. 5C). Due to the slow reaction dynamics and low concentrations of the “PCB” sample LED flash photolysis was used to monitor the dynamic processes. Samples were excited with a 10-ms, 455-nm LED flash in a UV-visible spectrophotometer to give the raw difference spectra shown in Fig. 6A. However, it is apparent that there were overlapping signals in the dataset, which originated from both the forward and reverse photoreactions of the PVB chromophore. Subtraction of scaled datasets collected on the same wavelength and time domains for the photoconversion of PVB from Pb to Pg (shown in Fig. 4A) and for the photoconversion of PVB from Pg to Pb (shown in Fig. 6B) resulted in the difference spectra shown in Fig. 7A, which should include components only from the PCB Pb to Pr photoconversion. Initial spectra show ground state bleaches at ∼325, 450, and 640 nm, and these spectra evolve over several seconds to a spectrum with a large bleach at ∼435 nm and a positive feature at ∼590 nm.

Bottom Line: The CBCR Tlr0924 contains both phycocyanobilin and phycoviolobilin chromophores, and these two species photoisomerize in parallel via spectrally and kinetically equivalent intermediates before the second step of the photoreaction where the reaction pathways diverge, the loss of a thioether linkage to a conserved cysteine residue occurs, and the phycocyanobilin reaction terminates in a red-absorbing state, whereas the phycoviolobilin reaction proceeds more rapidly to a final green-absorbing state.Our analysis shows that Tlr0924 undergoes an unprecedented long photoreaction that spans from picoseconds to seconds.We show that the thermally driven, long timescale changes are less complex than those reported for the red/far-red photocycles of the related phytochrome photoreceptors.

View Article: PubMed Central - PubMed

Affiliation: From the Manchester Institute of Biotechnology and Photon Science Institute, Faculty of Life Sciences, The University of Manchester, Manchester M13 9PL, United Kingdom and.

Show MeSH
Related in: MedlinePlus