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Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl
C Fernández1, K Adeishvili, K Wüthrich
1Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zurich, Switzerland.
Abstract:
The (2)H,(13)C,(15)N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30 degrees C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The (13)C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.
Insights
Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine the structure of the OmpX protein from Escherichia coli in solution. This study demonstrates NMR
Area of Science:
- Structural biology
- Biochemistry
- Molecular biology
Background:
- Integral membrane proteins play crucial roles in cellular functions.
- Determining the structure of membrane proteins in a native-like environment is challenging.
- OmpX is a model outer membrane protein from Escherichia coli.
Purpose of the Study:
- To determine the solution structure of the OmpX protein.
- To utilize Nuclear Magnetic Resonance (NMR) spectroscopy for structural elucidation.
- To demonstrate the utility of relaxation-optimized NMR techniques for membrane protein studies.
Main Methods:
- Reconstitution of the (2H,13C,15N)-labeled OmpX protein in dihexanoyl phosphatidylcholine (DHPC) mixed micelles.
- Acquisition of Transverse Relaxation-Optimized Spectroscopy (TROSY)-type triple resonance NMR experiments.
- Recording of TROSY-type Nuclear Overhauser Effect (NOE) spectra.
Main Results:
- Complete sequence-specific NMR assignments for the OmpX polypeptide backbone were obtained.
- Identification of regular secondary structure elements (alpha-helices and beta-sheets) in solution.
- Conformational constraints were collected for global fold computation, revealing similarity to crystal structure.
Conclusions:
- The solution structure of OmpX determined by NMR is consistent with its crystal structure.
- Relaxation-optimized NMR techniques are effective for studying integral membrane proteins.
- This work provides a foundation for further structural refinement and functional studies of OmpX.