An evaluation of detergents for NMR structural studies of membrane proteins.
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 (1)H-(15)N 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 alpha and beta 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 alpha-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 beta subunits form native-like complexes with bacteriochlorophyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of (15)N transverse, longitudinal and (15)N[(1)H] 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.
Krueger-Koplin
Ray D
RD
Biochemistry Department, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, U.S.A.
Sorgen
Paul L
PL
Krueger-Koplin
Suzanne T
ST
Rivera-Torres
Iván O
IO
Cahill
Sean M
SM
Hicks
David B
DB
Grinius
Leo
L
Krulwich
Terry A
TA
Girvin
Mark E
ME
D017920:Antiporters / Q000737:chemistry / Q000494:pharmacology; D003902:Detergents / Q000737:chemistry* / Q000494:pharmacology; D029968:Escherichia coli Proteins; D005989:Glycerides / Q000737:chemistry* / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry* / Q000187:drug effects / Q000494:pharmacology; D008823:Micelles; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry / Q000187:drug effects; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D006180:Proton-Translocating ATPases / Q000737:chemistry; D013113:Spin Labels
C492788:1-palmitoyl-2-hydroxy-sn-glycero-3-(phospho-RAC-(1-glycerol)); D017920:Antiporters; D003902:Detergents; D029968:Escherichia coli Proteins; D005989:Glycerides; D008565:Membrane Proteins; D008823:Micelles; D013113:Spin Labels; C075436:EmrE protein, E coli; C103607:mitochondrial ATPase subunit c; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases