NMR characterization of membrane protein-detergent micelle solutions by use of microcoil equipment.
Using microcoil NMR technology, the uniformly (2)H,(15)N-labeled integral membrane protein OmpX, and the phosphocholine derivative detergent Fos-10 (n-decylphosphocholine), we investigated solutions of mixed protein-detergent micelles to determine the influence of the detergent concentration on the NMR spectra of the protein. In a first step, we identified key parameters that influence the composition of the micelle solutions, which resulted in a new protocol for the preparation of well-defined concentrated protein solutions. This led to the observation that high-quality 2D [(15)N,(1)H]-transverse relaxation-optimized spectroscopy (TROSY) spectra of OmpX reconstituted in mixed micelles with Fos-10 were obtained only in a limited range of detergent concentrations. Outside of this range from about 90-180 mM, we observed a significant decrease of the average peak intensity. Relaxation-optimized NMR measurements of the rotational and translational diffusion coefficients of the OmpX/Fos-10 mixed micelles, D(r) and D(t), respectively, then showed that the stoichiometry and the effective hydrodynamic radius of the protein-containing micelles are not significantly affected by high Fos-10 concentrations and that the deterioration of NMR spectra is due to the increased viscosity at high detergent concentrations. The paper thus provides a basis for refined guidelines on the preparation of integral membrane proteins for structural studies.
Stanczak
Pawel
P
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Horst
Reto
R
Serrano
Pedro
P
Wüthrich
Kurt
K
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents / Q000737:chemistry*; D003903:Deuterium; D029968:Escherichia coli Proteins; D006867:Hydrolases; D009682:Magnetic Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D012996:Solutions
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008823:Micelles; D009587:Nitrogen Isotopes; D012996:Solutions; C482905:OmpX protein, E coli; D003903:Deuterium; D006867:Hydrolases