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J Am Chem Soc. 2009 Dec 30;131(51):18450-6. doi: 10.1021/ja907842u.

NMR characterization of membrane protein-detergent micelle solutions by use of microcoil equipment.

Author information

1
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Abstract

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.

PMID:
19950959
PMCID:
PMC2893019
DOI:
10.1021/ja907842u
[Indexed for MEDLINE]
Free PMC Article

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