Department of Biochemistry and Biophysics and Program in Cellular and Molecular Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Biophys J. 2011 Sep 7;101(5):1095-104. doi: 10.1016/j.bpj.2011.07.031.
While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events.
虽然病毒融合肽（例如血凝素 (HA) 和 gp41）在病毒-细胞膜融合中的重要性已得到确立，但尚不清楚这些肽如何增强膜融合，特别是在低肽/脂质比下，这些肽不会导致膜裂解。我们检测了野生型 HA 融合肽及其两个突变体 G1E 和 G13L 对 1,2-二油酰基-3- sn -磷酸胆碱/1,2-二油酰基-3- sn -磷酸乙醇胺/鞘磷脂/胆固醇（35:30:15:20）膜的双层结构、它们在脂质双层中的结构以及它们对膜融合的影响。所有肽都与高度弯曲的囊泡结合，但只有在聚乙二醇存在的情况下才会触发融合。在低（1:200）肽/脂质比下，野生型肽显著增强了内容物混合的程度和这些过程的速率常数，并显著增强了双层内部堆积并填充了膜自由体积。突变体没有引起内容物混合或内部堆积的变化。圆二色性、偏振衰减全内反射傅里叶变换红外光谱测量和膜扰动测量都符合野生型 HA 肽结构的倒 V 模型。类似的测量表明，G13L 突变体采用了一种较少螺旋的构象，其中 N 端更接近双层界面，从而破坏了 V 结构。G1E 肽几乎不会扰动双层，可能位于界面上方稍远处。融合测量表明，野生型肽通过其占据跨膜接触结构中疏水区的能力，促进了茎向扩展的跨膜接触中间体的转化。虽然野生型肽增加了初始中间体和最终孔形成的速率，但我们的结果并没有说明这些效应的机制，但它们确实为这些事件的过渡态的稳定性留下了可能性。
