Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain.
Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [(15)N,(1)H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [(15)N,(1)H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.
Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, Switzerland.
Christos
C
Tzitzilonis
Cédric
C
Eichmann
Innokentiy
I
Maslennikov
Senyon
S
Choe
Roland
R
Riek
D032701:Biomimetics; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C530142:YgaP protein, E coli