Yang Yu, Yao Hongwei, Hong Mei
Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States.
J Phys Chem B. 2015 Apr 16;119(15):4993-5001. doi: 10.1021/acs.jpcb.5b01001. Epub 2015 Apr 7.
Nonlamellar lipid membranes are frequently induced by proteins that fuse, bend, and cut membranes. Understanding the mechanism of action of these proteins requires the elucidation of the membrane morphologies that they induce. While hexagonal phases and lamellar phases are readily identified by their characteristic solid-state NMR line shapes, bicontinuous lipid cubic phases are more difficult to discern, since the static NMR spectra of cubic-phase lipids consist of an isotropic (31)P or (2)H peak, indistinguishable from the spectra of isotropic membrane morphologies such as micelles and small vesicles. To date, small-angle X-ray scattering is the only method to identify bicontinuous lipid cubic phases. To explore unique NMR signatures of lipid cubic phases, we first describe the orientation distribution of lipid molecules in cubic phases and simulate the static (31)P chemical shift line shapes of oriented cubic-phase membranes in the limit of slow lateral diffusion. We then show that (31)P T2 relaxation times differ significantly between isotropic micelles and cubic-phase membranes: the latter exhibit 2 orders of magnitude shorter T2 relaxation times. These differences are explained by the different time scales of lipid lateral diffusion on the cubic-phase surface versus the time scales of micelle tumbling. Using this relaxation NMR approach, we investigated a DOPE membrane containing the transmembrane domain (TMD) of a viral fusion protein. The static (31)P spectrum of DOPE shows an isotropic peak, whose T2 relaxation times correspond to that of a cubic phase. Thus, the viral fusion protein TMD induces negative Gaussian curvature, which is an intrinsic characteristic of cubic phases, to the DOPE membrane. This curvature induction has important implications to the mechanism of virus-cell fusion. This study establishes a simple NMR diagnostic probe of lipid cubic phases, which is expected to be useful for studying many protein-induced membrane remodeling phenomena in biology.
非层状脂质膜常常由能够融合、弯曲和切割膜的蛋白质诱导形成。要理解这些蛋白质的作用机制，需要阐明它们所诱导的膜形态。虽然六方相和层状相可以通过其特征性的固态核磁共振线形很容易地识别出来，但双连续脂质立方相则更难辨别，因为立方相脂质的静态核磁共振谱由一个各向同性的（31）P或（2）H峰组成，这与诸如胶束和小囊泡等各向同性膜形态的谱无法区分。到目前为止，小角X射线散射是识别双连续脂质立方相的唯一方法。为了探索脂质立方相独特的核磁共振特征，我们首先描述了脂质分子在立方相中的取向分布，并在横向扩散缓慢的极限情况下模拟了取向立方相膜的静态（31）P化学位移线形。然后我们表明，各向同性胶束和立方相膜之间的（31）P T2弛豫时间有显著差异：后者的T2弛豫时间短2个数量级。这些差异可以通过立方相表面脂质横向扩散的时间尺度与胶束翻滚的时间尺度不同来解释。利用这种弛豫核磁共振方法，我们研究了一种含有病毒融合蛋白跨膜结构域（TMD）的DOPE膜。DOPE的静态（31）P谱显示一个各向同性峰，其T2弛豫时间与立方相的一致。因此，病毒融合蛋白TMD会给DOPE膜诱导出负高斯曲率，这是立方相的一个固有特征。这种曲率诱导对病毒-细胞融合机制具有重要意义。本研究建立了一种简单的脂质立方相核磁共振诊断探针，有望用于研究生物学中许多蛋白质诱导的膜重塑现象。
