Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Biomol NMR. 2012 Oct;54(2):129-33. doi: 10.1007/s10858-012-9658-x. Epub 2012 Aug 14.
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.
采用微线圈 NMR 监测了整体膜蛋白（IMP）OmpW 的水溶液的优化，该方法仅使用微克级的蛋白质和去污剂即可获得 NMR 谱。发现去污剂 30-Fos（2-十一烷基磷酸胆碱）可产生最佳的二维 [(15)N，(1)H]-TROSY 相关 NMR 谱 [(2)H，(15)N]-标记的 OmpW。然后，我们针对 OmpW 结构测定优化了 30-Fos 浓度、样品温度和长时间稳定性以及蛋白质的氘化水平。讨论了一些用于结构生物学中β桶整体膜蛋白重建的新准则。
