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Redox-dependent dynamics of putidaredoxin characterized by amide proton exchange
T A Lyons1, G Ratnaswamy, T C Pochapsky
1Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Protein Science : a Publication of the Protein Society
|April 1, 1996
Summary
This study used multidimensional NMR to map protein dynamics in oxidized and reduced putidaredoxin (Pdx). The oxidized form shows faster amide proton exchange, particularly near the metal center, impacting its interaction with cytochrome P-450cam.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Putidaredoxin (Pdx) is an Fe2S2 ferredoxin crucial for cytochrome P-450cam (CYP101) reduction.
- Previous NMR studies established a solution structure model for oxidized Pdx and described redox-dependent proton NMR features.
Purpose of the Study:
- To assign 15N resonances for amide and side-chain nitrogens in both oxidized and reduced Pdx.
- To characterize local protein dynamics in Pdx across different oxidation states using amide proton exchange rates.
Main Methods:
- Multidimensional NMR techniques, including NOESY-(1H/15N) HMQC and TOCSY-(1H/15N) HSQC, were employed.
- 15N resonance assignments were achieved using uniformly 15N-labeled Pdx samples.
- Residue-specific amide proton exchange rates were measured via saturation transfer and H2O/D2O exchange methods.
Main Results:
- 15N resonance assignments were successfully obtained for both oxidized and reduced Pdx.
- Amide proton exchange rates generally increased in the oxidized state compared to the reduced state.
- Significant oxidation-state dependence in exchange rates was observed for residues near the metal center and in compact structural regions.
Conclusions:
- Oxidation state significantly influences the local dynamics of putidaredoxin, particularly in regions critical for protein-protein interactions.
- These dynamic changes are relevant to the known redox-dependent binding interaction between Pdx and cytochrome P-450cam (CYP101).