Long-Distance Protonation-Conformation Coupling in Phytochrome Species
Abstract
:1. Introduction
2. Results
2.1. Protein Variants, Their Spectroscopic Characterization and Fluorescein Labeling of the Photosensory Module
2.2. pH-Dependence of UV–Vis Absorption of Agp1-PGP and Its Variants
2.3. pH-Dependence of Conformational Dynamics and Structural Constraints in Cph1 Constructs Using Time-Resolved Fluorescence Anisotropy
3. Discussion
4. Materials and Methods
4.1. Phytochrome Mutagenese, Expression and Purification
4.2. Phytochrome Labeling with IAF
4.3. UV–Vis Spectroscopy, pH-Titration and pKa Determination
4.4. Time-Resolved Fluorescence Spectroscopy
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Sample | Chromophore pKDPC | pKDPC a |
---|---|---|
Agp1 PGP WT | 10.70 ± 0.02 | - |
Agp1 PGP C295S | 10.70 ± 0.01 | 0 ± 0.03 |
Agp1 PGP C279S | 10.40 ± 0.02 | −0.3 ± 0.03 |
Agp1 PGP C279S/C295S | 10.51 ± 0.02 | −0.19 ± 0.04 |
Agp1 PGP C279S/C295S/V364C | 10.50 ± 0.02 | −0.20 ± 0.04 |
Agp1 PGP WT-AF | 10.42 ± 0.02 | −0.28 ± 0.04 |
Agp1 PGP C295S/C279-AF | 10.49 ± 0.02 | −0.21 ± 0.04 |
Agp1 PGP C279S/C295-AF | 10.45 ± 0.03 | −0.25 ± 0.05 |
Agp1 PGP C279S/C295S/V364C-AF | 10.60 ± 0.03 | −0.09 ± 0.05 |
(A) Agp1 PGP WT-AF | ||||||||
pH | r0 | ϕ1 (ns) | ϕ2 (ns) | ϕ3 (ns) a | β1 | β2 | β3 | χred2 |
7.4 | 0.35 | 0.10 | 1.05 | 30.0 | 0.052 | 0.065 | 0.232 | 0.95 |
8.0 | 0.35 | 0.08 | 0.82 | 30.0 | 0.063 | 0.066 | 0.220 | 0.93 |
8.6 | 0.34 | 0.08 | 0.82 | 30.0 | 0.058 | 0.071 | 0.211 | 0.93 |
9.0 | 0.34 | 0.11 | 0.91 | 30.0 | 0.066 | 0.066 | 0.207 | 0.92 |
9.5 | 0.34 | 0.07 | 0.87 | 30.0 | 0.062 | 0.076 | 0.202 | 1.00 |
10.2 | 0.33 | 0.13 | 0.92 | 30.0 | 0.059 | 0.079 | 0.192 | 0.93 |
(B) Agp1 PGP C279-AF | ||||||||
pH | r0 | ϕ1 (ns) | ϕ2 (ns) | ϕ3 (ns) a | β1 | β2 | β3 | χred2 |
7 | 0.34 | 0.10 | 1.10 | 30 | 0.048 | 0.078 | 0.214 | 0.96 |
7.5 | 0.34 | 0.31 | 2.25 | 30 | 0.070 | 0.071 | 0.199 | 1.04 |
9.1 | 0.34 | 0.23 | 1.17 | 30 | 0.078 | 0.082 | 0.180 | 0.87 |
9.5 | 0.34 | 0.17 | 1.10 | 30 | 0.065 | 0.098 | 0.174 | 1.06 |
10.3 | 0.34 | 0.18 | 1.17 | 30 | 0.077 | 0.097 | 0.161 | 1.02 |
10.8 | 0.34 | 0.27 | 1.5 | 30 | 0.088 | 0.093 | 0.160 | 0.96 |
(C) Agp1 PGP C295-AF | ||||||||
pH | r0 | ϕ1 (ns) | ϕ2 (ns) | ϕ3 (ns) a | β1 | β2 | β3 | χred2 |
7.5 | 0.34 | 0.04 | 0.50 | 30 | 0.054 | 0.055 | 0.231 | 1.01 |
7.8 | 0.34 | 0.04 | 0.41 | 30 | 0.049 | 0.056 | 0.235 | 0.96 |
8.0 | 0.34 | 0.04 | 0.50 | 30 | 0.056 | 0.054 | 0.229 | 0.95 |
8.6 | 0.34 | 0.05 | 0.51 | 30 | 0.049 | 0.056 | 0.235 | 0.95 |
9.2 | 0.34 | 0.04 | 0.52 | 30 | 0.058 | 0.058 | 0.224 | 1.01 |
10.0 | 0.34 | 0.06 | 0.60 | 30 | 0.060 | 0.060 | 0.2196 | 0.96 |
10.5 | 0.34 | 0.07 | 0.70 | 30 | 0.060 | 0.063 | 0.2162 | 0.98 |
11.0 | 0.34 | 0.05 | 0.60 | 30 | 0.061 | 0.066 | 0.2135 | 0.94 |
(D) Agp1 PGP V364C-AF | ||||||||
pH | r0 | ϕ1 (ns) | ϕ2 (ns) | ϕ3 (ns) a | β1 | β2 | β3 | χred2 |
6.3 | 0.34 | 0.15 | 1.40 | 30 | 0.049 | 0.086 | 0.204 | 0.96 |
7.5 | 0.34 | 0.15 | 1.40 | 30 | 0.059 | 0.085 | 0.199 | 0.94 |
8.9 | 0.34 | 0.15 | 1.35 | 30 | 0.065 | 0.088 | 0.187 | 1.01 |
9.5 | 0.34 | 0.15 | 0.60 | 30 | 0.076 | 0.089 | 0.174 | 0.97 |
10.0 | 0.34 | 0.15 | 1.70 | 30 | 0.080 | 0.088 | 0.171 | 0.98 |
Sample | Chromophore pKDPC | pKa of Conformational Change |
---|---|---|
Agp1 PGP WT-AF (labeling in GAF) | 10.42 ± 0.02 | 9.0 ± 0.2 |
Agp1 PGP C295S/C279-AF (labeling in β-sheet of GAF) | 10.49 ± 0.02 | 9.2 ± 0.1 |
Agp1 PGP C279S/C295-AF (labeling in long helix of GAF) | 10.45 ± 0.03 | 9.7 ± 0.2 |
Agp1 PGP C279S/C295S/V364C-AF (labeling in β-sheet of PHY) | 10.60 ± 0.03 | 9.0 ± 0.2 |
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Sadeghi, M.; Balke, J.; Rafaluk-Mohr, T.; Alexiev, U. Long-Distance Protonation-Conformation Coupling in Phytochrome Species. Molecules 2022, 27, 8395. https://doi.org/10.3390/molecules27238395
Sadeghi M, Balke J, Rafaluk-Mohr T, Alexiev U. Long-Distance Protonation-Conformation Coupling in Phytochrome Species. Molecules. 2022; 27(23):8395. https://doi.org/10.3390/molecules27238395
Chicago/Turabian StyleSadeghi, Maryam, Jens Balke, Timm Rafaluk-Mohr, and Ulrike Alexiev. 2022. "Long-Distance Protonation-Conformation Coupling in Phytochrome Species" Molecules 27, no. 23: 8395. https://doi.org/10.3390/molecules27238395