Sanders C R
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.
Biophys J. 1993 Jan;64(1):171-81. doi: 10.1016/S0006-3495(93)81352-3.
The direct measurement of 13C chemical shift anisotropies (CSA) and 31P-13C dipolar splitting in random dispersions of unlabeled L alpha-phase phosphatidylcholine (PC) has traditionally been difficult because of extreme spectral boradening due to anisotropy. In this study, mixtures of dimyristoyl phosphatidylcholine (DMPC) with three different detergents known to promote the magnetic orientation of DMPC were employed to eliminate the powder-pattern nature of signals without totally averaging out spectral anisotropy. The detergents utilized were CHAPSO, Triton X-100, and dihexanoylphosphatidylcholine (DHPC). Using such mixtures, many of the individual 13C resonances from DMPC were resolved and a number of 13C-31P dipolar couplings were evident. In addition, differing line widths were observed for the components of some dipolar doublets, suggestive of dipolar/chemical shift anisotropy (CSA) relaxation interference effects. Oriented sample resonance assignments were made by varying the CHAPSO or DHPC to DMPC ratio to systematically scale overall bilayer order towards the isotropic limit. In this manner, peaks could be identified based upon extrapolation to their isotropic positions, for which assignments have previously been made (Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355-358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821-3849). It was observed that the plots of CSA or dipolar coupling versus overall bilayer order obtained from DHPC and CHAPSO titrations were linear. Estimates of the intrinsic dipolar couplings and chemical shift anisotropies for pure DMPC bilayers were made by extrapolating shifts and couplings from the detergent titrations to zero detergent. Both detergent titrations led to similar "intrinsic" CSAs and dipolar couplings. Results extracted from an oriented Triton-DMPC mixture also led to similar estimates for the detergent-free DMPC shifts and couplings. The results from these experiments were found to compare favorably with limited measurements made from pure L alpha PC. This detergent-based method for assigning spectra and for determining dipolar couplings and CSA in detergent-free systems should be extendable to other lipid systems. The resulting data set from this study may prove useful in future modeling of the structure and dynamics of DMPC bilayers. In addition, the fact that experiments utilizing each of the three detergents led to similar estimates for the spectral parameters of pure DMPC, and the fact that spectral parameter versus bilayer order plots were linear, indicate that the averaged conformation and dynamics of DMPC in the presence of the three detergents are very similar to those of pure L alpha DMPC.
传统上，由于各向异性导致的极端谱线展宽，对未标记的Lα相磷脂酰胆碱（PC）随机分散体中的13C化学位移各向异性（CSA）和31P-13C偶极分裂进行直接测量一直很困难。在本研究中，使用二肉豆蔻酰磷脂酰胆碱（DMPC）与三种已知可促进DMPC磁取向的不同去污剂的混合物，以消除信号的粉末状图谱性质，同时又不完全消除谱线各向异性。所使用的去污剂为CHAPSO、Triton X-100和二己酰磷脂酰胆碱（DHPC）。使用这些混合物，DMPC的许多单个13C共振得以分辨，并且一些13C-31P偶极耦合很明显。此外，观察到一些偶极双峰的组分具有不同的线宽，这表明存在偶极/化学位移各向异性（CSA）弛豫干扰效应。通过改变CHAPSO或DHPC与DMPC的比例，使整体双层有序性系统地向各向同性极限变化，从而对取向样品的共振进行归属。通过这种方式，可以根据外推到其各向同性位置来识别峰，此前已对这些位置进行了归属（Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355 - 358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821 - 3849）。观察到从DHPC和CHAPSO滴定得到的CSA或偶极耦合与整体双层有序性的关系图是线性的。通过将去污剂滴定中的位移和耦合外推到零去污剂，对纯DMPC双层的固有偶极耦合和化学位移各向异性进行了估计。两种去污剂滴定都得到了类似的“固有”CSA和偶极耦合。从取向的Triton-DMPC混合物中提取的结果也对无去污剂的DMPC位移和耦合给出了类似的估计。发现这些实验的结果与从纯Lα PC进行的有限测量结果相比具有优势。这种基于去污剂的用于在无去污剂系统中进行光谱归属以及确定偶极耦合和CSA的方法应该可以扩展到其他脂质系统。本研究得到的数据集可能在未来DMPC双层的结构和动力学建模中证明是有用的。此外，使用三种去污剂中的每一种进行的实验都对纯DMPC的光谱参数给出了类似的估计，以及光谱参数与双层有序性的关系图是线性的这一事实，表明在三种去污剂存在下DMPC的平均构象和动力学与纯Lα DMPC的非常相似。
