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Combining NMR and EPR methods for homodimer protein structure determination
Yunhuang Yang1, Theresa A Ramelot, Robert M McCarrick
1Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
Journal of the American Chemical Society
|August 12, 2010
Summary
This study integrates paramagnetic relaxation enhancements (PRE) and double electron electron resonance (DEER) with nuclear magnetic resonance (NMR) to determine protein complex structures. This combined approach accurately defines homodimer structures, even for large molecules.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Developing robust methods for characterizing large protein complexes using solution-state NMR is crucial.
- Integrating complementary techniques enhances structural determination capabilities.
- Paramagnetic relaxation enhancements (PRE) and electron paramagnetic resonance (EPR) techniques like double electron electron resonance (DEER) offer valuable long-range distance constraints (10-70 Å).
Purpose of the Study:
- To integrate PRE and DEER data with conventional solution-state NMR for structural characterization.
- To determine the structure of the homodimer Dsy0195 from Desulfitobacterium hafniense.
- To assess the sufficiency of combined NMR, PRE, and DEER restraints for automated structure determination.
Main Methods:
- Utilized conventional solution-state nuclear magnetic resonance (NMR) methods.
- Incorporated distance constraints from paramagnetic relaxation enhancements (PRE).
- Integrated distance constraints from double electron electron resonance (DEER), an electron paramagnetic resonance (EPR) technique.
Main Results:
- The integration of conventional NMR with a few DEER and PRE constraints enabled accurate structure determination of the Dsy0195 homodimer.
- The automatic NMR-based structure determination program CYANA successfully built a network of interchain nuclear Overhauser effect constraints.
- The defined homodimer interface and global structure were accurately determined.
- DEER distance constraints have minimal upper molecular weight limitations.
- PRE constraint utility is limited by the accuracy of protein chemical shift assignments.
Conclusions:
- Combining conventional NMR with PRE and DEER provides a powerful and robust method for structural characterization of homodimers and multiprotein complexes.
- This integrated approach facilitates accurate definition of protein interfaces and global structures.
- The method is highly scalable, with DEER offering advantages for large molecular weight complexes.
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