Epand R M, Epand R F, Martin I, Ruysschaert J M
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
Biochemistry. 2001 Jul 31;40(30):8800-7. doi: 10.1021/bi0107187.
We have studied a group of fusion peptides of influenza hemagglutinin in which the N-terminal amino acid, Gly (found in the wild-type peptide), has been systematically substituted with Ala, Ser, Val, or Glu. The activity of the intact hemagglutinin protein with these same substitutions has already been reported. As a measure of the extent of modulation of intrinsic membrane curvature by these peptides, we determined their effects on the polymorphic phase transition of dipalmitoleoylphosphatidylethanolamine. The wild-type peptide is the only one that, at pH 5, can substantially decrease the temperature of this transition. This is also the only form in which the intact protein promotes contents mixing in cells. The Ala and Ser mutant hemagglutinins exhibit a hemifusion phenotype, and their fusion peptides have little effect on lipid polymorphism at low pH. The two mutant proteins that are completely fusion inactive are the Val and Glu mutant hemagglutinins. The fusion peptides from these forms significantly increase the polymorphic phase transition temperature at low pH. We find that the effect of the fusion peptides on membrane curvature, as monitored by a shift in the temperature of this polymorphic phase transition, correlates better with the fusogenic activities of the corresponding protein than do measurements of the isotropic (31)P NMR signals or the ability to induce the fusion of liposomes. The inactivity of the hemagglutinin protein with the hydrophobic Val mutation can be explained by the change in the angle of membrane insertion of the helical fusion peptide as measured by polarized FTIR. Thus, the nature of the interactions of the fusion peptides with membranes can, in large part, explain the differences in the fusogenic activity of the intact protein.
我们研究了一组流感血凝素融合肽，其中野生型肽中的N端氨基酸甘氨酸已被系统地替换为丙氨酸、丝氨酸、缬氨酸或谷氨酸。具有相同替换的完整血凝素蛋白的活性已有报道。作为这些肽对内在膜曲率调节程度的一种衡量，我们测定了它们对二棕榈酰油酰磷脂酰乙醇胺多晶型相变的影响。野生型肽是唯一在pH 5时能大幅降低该相变温度的肽。这也是完整蛋白促进细胞内内容物混合的唯一形式。丙氨酸和丝氨酸突变型血凝素表现出半融合表型，它们的融合肽在低pH下对脂质多态性几乎没有影响。完全无融合活性的两种突变蛋白是缬氨酸和谷氨酸突变型血凝素。这些形式的融合肽在低pH下显著提高多晶型相变温度。我们发现，通过该多晶型相变温度的变化监测，融合肽对膜曲率的影响与相应蛋白的融合活性的相关性，比各向同性（31）P NMR信号的测量或诱导脂质体融合的能力更好。通过偏振傅里叶变换红外光谱测量，具有疏水缬氨酸突变的血凝素蛋白的无活性可以用螺旋融合肽膜插入角度的变化来解释。因此，融合肽与膜相互作用的性质在很大程度上可以解释完整蛋白融合活性的差异。
