NMR Characterization of Membrane Protein−Detergent Micelle Solutions by Use of Microcoil Equipment

• Published in: Journal of the American Chemical Society Vol. 131; no. 51; pp. 18450 - 18456
• Main Authors: Stanczak, Pawel, Horst, Reto, Serrano, Pedro, Wüthrich, Kurt
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30.12.2009
• Subjects: Bacterial Outer Membrane Proteins; Detergents - chemistry; Deuterium; Escherichia coli Proteins; Hydrolases; Magnetic Resonance Spectroscopy - instrumentation; Magnetic Resonance Spectroscopy - methods; Membrane Proteins - chemistry; Micelles; Molecular Structure; Nitrogen Isotopes; Solutions
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja907842u

Using microcoil NMR technology, the uniformly 2H,15N-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 [15N,1H]-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.