Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA.
Biophys J. 2010 Nov 17;99(10):3255-63. doi: 10.1016/j.bpj.2010.09.028.
Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigid transmembrane (TM) β-barrel. PFO is believed to interact with liquid ordered lipid domains (lipid rafts). Because the origin of TM protein affinity for rafts is poorly understood, we investigated PFO raft affinity in vesicles having coexisting ordered and disordered lipid domains. Fluorescence resonance energy transfer (FRET) from PFO Trp to domain-localized acceptors indicated that PFO generally has a raft affinity between that of LW peptide (low raft affinity) and cholera toxin B (high raft affinity) in vesicles containing ordered domains rich in brain sphingomyelin or distearoylphosphatidylcholine. FRET also showed that ceramide, which increases exposure of cholesterol to water and thus displaces it from rafts, does not displace PFO from ordered domains. This can be explained by shielding of PFO-bound cholesterol from water. Finally, FRET showed that PFO affinity for ordered domains was higher in its non-TM (prepore) form than in its TM form, demonstrating that the TM portion of PFO interacts unfavorably with rafts. Microscopy studies in giant unilamellar vesicles confirmed that PFO exhibits intermediate raft affinity, and showed that TM PFO (but not non-TM PFO) concentrated at the edges of liquid ordered domains. These studies suggest that a combination of binding to raft-associating molecules and having a rigid TM structure that is unable to pack well in a highly ordered lipid environment can control TM protein domain localization. To accommodate these constraints, raft-associated TM proteins in cells may tend to locate within liquid disordered shells encapsulated within ordered domains.
当与胆固醇相互作用时，产气荚膜梭菌 α 毒素（PFO）插入膜中并形成刚性跨膜（TM）β桶。PFO 被认为与液态有序脂质域（脂质筏）相互作用。由于 TM 蛋白与筏的亲和力的起源理解得很差，我们研究了具有共存有序和无序脂质域的囊泡中 PFO 筏的亲和力。来自 PFO Trp 到域定位受体的荧光共振能量转移（FRET）表明，在富含脑鞘磷脂或二硬脂酰基磷脂酰胆碱的有序域的囊泡中，PFO 通常具有介于 LW 肽（低筏亲和力）和霍乱毒素 B（高筏亲和力）之间的筏亲和力。FRET 还表明，神经酰胺增加了胆固醇暴露在水中的程度，从而将其从筏中置换出来，但不会将 PFO 从有序域中置换出来。这可以用 PFO 结合的胆固醇被水屏蔽来解释。最后，FRET 表明，在其非 TM（前孔）形式中，PFO 对有序域的亲和力高于其 TM 形式，表明 PFO 的 TM 部分与筏的相互作用不利。在巨大的单层囊泡中的显微镜研究证实了 PFO 具有中间的筏亲和力，并表明 TM PFO（而非非 TM PFO）在液态有序域的边缘浓缩。这些研究表明，与筏结合的分子结合以及具有刚性 TM 结构，该结构不能在高度有序的脂质环境中很好地组装，可以控制 TM 蛋白结构域的定位。为了适应这些限制，细胞中与筏相关的 TM 蛋白可能倾向于位于有序域内封装的液态无序壳内。
