An evaluation of detergents for NMR structural studies of membrane proteins
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- 作者：Ray D. Krueger-Koplin ; Paul L. Sorgen ; Suzanne T. Krueger-Koplin ; Iv¨¢n O. Rivera-Torres ; Sean M. Cahill ; David B. Hicks ; Leo Grinius ; Terry A. Krulwich and Mark E. Girvin
- 关键词：detergent ; lysolipids ; membrane ; protein ; structure
- 刊名：Journal of Biomolecular NMR
- 出版年：2004
- 出版时间：2004
- 年：2004
- 卷：28
- 期：1
- 页码：43-57
- 全文大小：322 KB
文摘
Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D 1H-15N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested – R. sphaeroides LH1 and subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter – with 1, 2, or 4 membrane spanning -helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 subunits form native-like complexes with bacteriochlorphyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of 15N transverse, longitudinal and 15N{1H} nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15–20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.