Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya str., 117997 Moscow, Russia.
J Am Chem Soc. 2010 Apr 28;132(16):5628-9. doi: 10.1021/ja9097498.
The choice of a suitable detergent-based membrane mimetic is of crucial importance for high-resolution NMR studies of membrane proteins. The present report describes a new approach of detergent screening. It is based on the comparison of 2D (1)H,(15)N-correlation spectra of a protein in a membrane-bilayer "reference" medium and in "trial" detergent-based environments. The proposed "reference" medium is the lipid-protein nanodisc (LPN) representing nanoscale phospholipid bilayers wrapped around by apolipoprotein A-1. The set of zwitterionic (DPC, DMPC/DHPC), anionic (SDS, LMPG, LPPG), and weakly cationic (LDAO) detergent-based media was screened for their ability to represent the native structure of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP. The VSD/LPN complexes composed of saturated zwitterionic (DMPC), anionic (DMPG), or a mixture of unsaturated differently charged (POPC/DOPG, 3:1) lipids were used as reference. All assayed detergent media demonstrate similar CD spectra of the domain with a high level (approximately 60%) of overall helicity but different 2D NMR spectra. Using the reference spectrum of the VSD in LPN, we were able to choose the detergent composition in which the membrane-like structure of the VSD is preserved.
选择合适的基于洗涤剂的膜模拟物对于膜蛋白的高分辨率 NMR 研究至关重要。本报告描述了一种新的洗涤剂筛选方法。它基于比较膜双层“参考”介质和“试验”基于洗涤剂的环境中蛋白质的 2D（1）H，（15）N 相关谱。所提出的“参考”介质是脂质-蛋白纳米盘（LPN），代表由载脂蛋白 A-1 包裹的纳米尺度磷脂双层。筛选了一组两性离子（DPC、DMPC/DHPC）、阴离子（SDS、LMPG、LPPG）和弱阳离子（LDAO）基于洗涤剂的介质，以评估它们代表分离的古细菌钾通道 KvAP 的电压感应结构域（VSD）的能力。由饱和两性离子（DMPC）、阴离子（DMPG）或不同带电（POPC/DOPG，3:1）混合不饱和脂质组成的 VSD/LPN 复合物被用作参考。所有测定的洗涤剂介质均表现出与总体螺旋度约为 60%的域相似的 CD 光谱，但二维 NMR 光谱不同。使用 LPN 中 VSD 的参考光谱，我们能够选择保持 VSD 类似膜结构的洗涤剂组成。
