Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles.
Optimization of aqueous solutions of the integral membrane protein (IMP) OmpW for NMR structure determination has been monitored with micro-coil NMR, which enables the acquisition of NMR spectra using only micrograms of protein and detergent. The detergent 30-Fos (2-undecylphosphocholine) was found to yield the best 2D [(15)N, (1)H]-TROSY correlation NMR spectra of [(2)H, (15)N]-labeled OmpW. For the OmpW structure determination we then optimized the 30-Fos concentration, the sample temperature and long-time stability, and the deuteration level of the protein. Some emerging guidelines for reconstitution of β-barrel integral membrane proteins in structural biology are discussed.
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Pawel
P
Stanczak
Qinghai
Q
Zhang
Reto
R
Horst
Pedro
P
Serrano
Kurt
K
Wüthrich
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D003903:Deuterium; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008823:Micelles; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D011487:Protein Conformation; D055550:Protein Stability
C578185:2-undecylphosphocholine; D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008823:Micelles; C064035:ompW protein, E coli; D010767:Phosphorylcholine; D003903:Deuterium