期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 131, 期 51, 页码 18450-18456出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja907842u
关键词
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资金
- Joint Center for Innovative Membrane Protein Technologies (NIH) [P50 GM073197]
- Joint Center for Structural Genomics (NIH) [U54 GM074898]
Using microcoil NMR technology, the uniformly H-2,N-15-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 olutions. This led to the observation that high-quality 2D [N-15,H-1]-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.
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