Parente R A, Nir S, Szoka F C
Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143.
J Biol Chem. 1988 Apr 5;263(10):4724-30.
A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.
一种合成的、具有两性的30个氨基酸的肽，其主要重复单元为Glu-Ala-Leu-Ala（GALA），旨在模拟病毒融合蛋白的融合序列的行为。GALA是一种水溶性肽，在中性pH下具有非周期性构象，当pH降至5.0时，它会变成两性α-螺旋并与双层膜相互作用。荧光能量转移测量表明，GALA可诱导磷脂酰胆碱小单层囊泡之间的脂质混合，但不能诱导大单层囊泡之间的脂质混合。这种脂质混合仅在pH 5.0时发生，而在中性pH下不发生。与脂质混合同时，通过动态光散射和内部体积测定可知，囊泡直径从500 Å增加到750 Å再增加到1000 Å。GALA在pH 5.0时诱导小分子（分子量450）泄漏的速度太快，无法检测到内容物混合。然而，在相同条件下大分子（分子量4100）的保留表明囊泡融合正在发生。对于100/1的脂质/肽比例，所有囊泡仅融合一次，而对于50/1的比例，则形成更高阶的融合产物。质量作用模型能很好地模拟荧光强度增加的动力学，并得出聚集和融合的速率常数。随着脂质与肽的比例从100/1降至50/1，聚集和融合的速率常数均增加，表明GALA是囊泡融合的真正诱导剂。二价阳离子的存在可在pH 7.5时改变GALA的构象，但其对脂质混合活性影响很小。通过改变序列同时保持氨基酸组成不变或缩短序列对GALA进行修饰。这些肽没有任何脂质混合活性，也不会诱导囊泡大小增加。总之，这些结果表明，GALA诱导的磷脂酰胆碱小单层囊泡融合既需要肽长度大于16个氨基酸，也需要疏水残基的特定拓扑结构。
