Microscale NMR screening of new detergents for membrane protein structural biology.
The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized beta-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly (2)H, (15)N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [ (15)N, (1)H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.
Zhang
Qinghai
Q
Department of Molecular Biology, Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Horst
Reto
R
Geralt
Michael
M
Ma
Xingquan
X
Hong
Wen-Xu
WX
Finn
M G
MG
Stevens
Raymond C
RC
Wüthrich
Kurt
K
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D004586:Electrophoresis; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006867:Hydrolases / Q000737:chemistry*; D008823:Micelles; D008832:Microchemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D017510:Protein Folding
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008823:Micelles; D010767:Phosphorylcholine; C482905:OmpX protein, E coli; D006867:Hydrolases