Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents.
For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle. In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer-membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques. The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water-soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein-detergent interactions in the mixed micelles. The application of paramagnetic probes to the well-characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein-detergent micelles.
Hilty
Christian
C
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, 8093 Zürich, Switzerland.
Wider
Gerhard
G
Fernández
César
C
Wüthrich
Kurt
K
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006867:Hydrolases / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008958:Models, Molecular; D008968:Molecular Conformation; D010742:Phospholipid Ethers / Q000737:chemistry*; D017186:Titrimetry
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008823:Micelles; D010742:Phospholipid Ethers; C482905:OmpX protein, E coli; D006867:Hydrolases