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Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy
S Weber1, G Richter, E Schleicher
1Institute of Experimental Physics, Free University Berlin, 14195 Berlin, Germany. stefan.weber@physik.fu-berlin.de
Biophysical Journal
|July 21, 2001
Summary
Escherichia coli DNA photolyase undergoes structural changes upon binding a cyclobutane pyrimidine dimer (CPD). These changes, studied using EPR and ENDOR, reveal a significant distance between the CPD and the flavin adenine dinucleotide (FAD) cofactor.
Area of Science:
- Biochemistry
- Molecular Biology
- Spectroscopy
Background:
- DNA photolyase repairs cyclobutane pyrimidine dimers (CPDs), a common DNA lesion.
- The enzyme's mechanism involves the flavin adenine dinucleotide (FAD) cofactor.
- Understanding structural changes upon substrate binding is crucial for elucidating repair mechanisms.
Purpose of the Study:
- To investigate structural alterations in E. coli DNA photolyase upon CPD binding.
- To characterize the microenvironment of the FAD cofactor during the repair process.
- To correlate experimental findings with theoretical predictions.
Main Methods:
- Continuous-wave electron paramagnetic resonance (EPR) spectroscopy.
- Electron-nuclear double resonance (ENDOR) spectroscopy.
- Density functional theory (DFT) based molecular orbital calculations with a polarized continuum model.
Main Results:
- EPR/ENDOR data indicate a distance of at least 0.6 nm between the CPD and the FAD's isoalloxazine ring.
- Observed shifts in FAD proton hyperfine coupling constants suggest a more nonpolar binding site.
- These shifts are attributed to water displacement upon CPD docking.
Conclusions:
- CPD binding induces significant structural rearrangements in DNA photolyase.
- The FAD cofactor's environment becomes less polar after CPD docking.
- Experimental results align with computational predictions of enzyme-substrate interactions.