PDF(Sci-hub)
Abstract：
We previously used an orthogonal high-throughput screen to select peptides that spontaneously cross synthetic lipid bilayers without bilayer disruption. Many of the 12-residue spontaneous membrane translocating peptides (SMTPs) selected from the library contained a 5-residue consensus motif, LRLLR in positions 5-9. We hypothesized that the conserved motif could be a necessary and sufficient minimal motif for translocation. To test this and to explore the mechanism of spontaneous membrane translocation, we synthesized seven arginine placement variants of LRLLRWC and compared their membrane partitioning, translocation, and perturbation to one of the parent SMTPs, called \"TP2\". Several motif variant peptides translocate into synthetic vesicles with rates that are similar to TP2. However, the peptide containing the selected motif, LRLLRWC, was not the fastest; sequence context is also important for translocation efficiency. Although none of these peptides permeabilize bilayers, the motif peptides translocate faster at higher peptide to lipid ratios, suggesting that bilayer perturbation and/or cooperative interactions are important for their translocation. On the other hand, TP2 translocates slower as its concentration is increased, suggesting that TP2 translocates as a monomer and is inhibited by lateral interactions in the membrane. TP2 and the LRLLR motif peptide induce lipid translocation, suggesting that lipids chaperone them across the bilayer. The other motif peptides do not induce lipid flip-flop, suggesting an alternate mechanism. Concatenated motifs translocate slower than the motifs alone. Variants of TP2 with shorter and longer arginine side-chain analogs translocate slower than TP2. In summary, these results suggest that multiple patterns of leucine and arginine can support spontaneous membrane translocation, and that sequence context is important for the contribution of the motifs. Because motifs do not make simple, additive contributions to spontaneous translocation, rational engineering of novel SMTPs will remain difficult, providing even more reason to pursue SMTP discovery with synthetic molecular evolution.
摘要：
我们先前使用正交高通量筛选来选择自发穿过合成脂质双层而没有双层破坏的肽。从文库中选择的许多12个残基的自发膜易位肽（SMTP）包含5个残基的共有基序，LRLLR在5-9号位置。我们假设保守的基序可能是易位的必要且足够的最小基序。为了测试这一点并探索自发性膜易位的机制，我们合成了LRLLRWC的七个精氨酸放置变体，并比较了它们的膜分配，易位,以及对其中一个父SMTP的扰动，称为“TP2”。几种基序变体肽以与TP2相似的速率易位到合成囊泡中。然而,含有选定基序的肽，LRLLRWC,不是最快的；序列上下文对易位效率也很重要。虽然这些肽都不能渗透双层，在较高的肽与脂质比率下，基序肽易位更快，表明双层扰动和/或合作相互作用对它们的易位很重要。另一方面,TP2随着其浓度的增加而变慢，表明TP2作为单体易位，并被膜中的横向相互作用抑制。TP2和LRLLR基序肽诱导脂质易位，表明脂质在双层中陪伴它们。其他基序肽不诱导脂质翻转，建议另一种机制。串联的基序比单独的基序更慢。具有较短和较长精氨酸侧链类似物的TP2变体比TP2更慢易位。总之,这些结果表明，亮氨酸和精氨酸的多种模式可以支持自发的膜易位，序列上下文对于基序的贡献很重要。因为图案并不简单，对自发性易位的累加贡献，新型SMTP的合理工程将仍然困难，提供了更多的理由来追求合成分子进化的SMTP发现。
