study of proton transfer in escherichia coli photolyase meng zhang biophysics graduate program the...
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Study of Proton Transfer in Escherichia coli PhotolyaseMeng Zhang
Biophysics Graduate Program
The Ohio State University
June 21, 2013
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Introduction• Photolyase: Photoenzyme that utilizes blue light to repair UV-
damaged DNA which causes skin cancer.
• Flavin cofactor: The catalytic cofactor of photolyase, flavin adenine dinucleotide (FAD), has five redox states.
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UV-vis spectroscopic properties of wild type Photolyase• The four redox states: FAD, FAD⦁̶ , FADH⦁, and FADH ̶ have different
UV-vis absorption spectra.
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Redox conversions in EcPL mutants
• Site-directed mutagenesis• N378C (N5 mutant) • E363L • N378C/E363L
• In the presence of substrate, N378C/E363L can stabilize at the FAD ⦁ state under continuous white light for hours.
Proton channels are are eliminated completely.
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The proposed proton channel in Photolyase
• The proton relay channel through E363 and surface water to N378 and then FAD;
• The proton diffusion channel through the substrate binding pocket.
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Reaction kineticsFADox FAD•- FADH⦁ FADH-
k12
k21 k32
k23
k43
k34
WT
WTN378C
N378C with substrate
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All in unit of min-1
FADox FAD•- FADH⦁ FADH-k12
k21 k32
k23
k43
k34
k12 k21 k23 k32 k34 k43 k43 with O2
WT
pH7 5.8×10-4 0.15 0.31 3.3×10-4 0.013 <1.0×10-4 0.025
pH9 0.33 0.71 4.1×10-3 0.077 <1.0×10-4 8.2×10-3
pH7 with S <1.0×10-4 0.19 <1.0×10-4 2.5×10-4
pH9 with S <1.0×10-4 0.37 <1.0×10-4 5.6×10-3
N378C
pH7 6.7×10-4 0.088 0.050 >10 >10 <1.0×10-3 0.023
pH9 0.054 0.034 >10 >10 <1.0×10-4 0.046
pH7 with S 8.3×10-4 0.010 >10 <0.01
pH9 with S 2.2×10-4 6.3×10-4 >10 <0.01
E363L
pH7 9.7×10-4 0.015 0.018 <1.0×10-4 >1
pH9 0.021 0.032 <1.0×10-4 >1
pH7 with S <1.0×10-4 0.031 <1.0×10-4 0.012
N378C E363L
pH7 0.026 4.4×10-3 >10 >10 7.3×10-3
Reaction kinetics
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Proton transfer in (6-4) Photolyase and Cryptochromes• (6-4) Photolyase
• Repairs (6-4) dipyrimidine photoproducts.
• Similar proton transfer pathway.
• Mutant N402C/E391L can stabilizes at FAD ⦁ state in the presence of substrate.
• Cryptochromes (ongoing work)
• Insect Cryptochrome• Plant Cryptochrome
C-terminal domain
FAD
PHR domain
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Conclusions
• N378 and E363 are the key residues for EcPL proton transfer.
• The proton channels in photolyase was proposed for the first time. Proton can be transported to flavin through two pathways: the proton relay channel (through E363 and N378 in EcPL); and the proton diffusion channel through the substrate binding pocket.
• Redox potentials of different FAD states are governed by the N5 residue and local environment.
• The proton transfer kinetics are critical to the mechanistic and functional divergence of Photolyase and Cryptochrome.
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Acknowledgements
Prof. Dongping ZhongProf. Aziz Sancar (UNC)
Ms. Lijuan WangDr. Jiang LiZheyun LiuChuang Tan
And all other colleagues in the Zhong group!
Funding from: NIH
Thank you!