Fuselier Taylor, Wimley William C
Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, Louisiana.
Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, Louisiana.
Biophys J. 2017 Aug 22;113(4):835-846. doi: 10.1016/j.bpj.2017.06.070.
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.
我们之前使用正交高通量筛选来选择能够自发穿过合成脂质双层且不破坏双层结构的肽段。从文库中选出的许多12个残基的自发膜转运肽（SMTPs）都含有一个5个残基的共有基序，位于第5至9位的LRLLR。我们推测这个保守基序可能是转运所必需且足够的最小基序。为了验证这一点并探索自发膜转运的机制，我们合成了LRLLRWC的7种精氨酸位置变体，并将它们的膜分配、转运和对一种称为“TP2”的亲本SMTP的扰动与TP2进行了比较。几种基序变体肽以与TP2相似的速率转运到合成囊泡中。然而，含有所选基序的肽LRLLRWC并不是最快的；序列背景对转运效率也很重要。尽管这些肽都不会使双层膜通透，但基序肽在较高的肽与脂质比例下转运得更快，这表明双层膜扰动和/或协同相互作用对它们的转运很重要。另一方面，TP2随着浓度增加转运得更慢，这表明TP2以单体形式转运并受到膜中侧向相互作用的抑制。TP2和LRLLR基序肽会诱导脂质转运，这表明脂质将它们护送穿过双层膜。其他基序肽不会诱导脂质翻转，这表明存在另一种机制。串联的基序比单独的基序转运得更慢。具有更短和更长精氨酸侧链类似物的TP2变体比TP2转运得更慢。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膜转运，并且序列背景对基序的贡献很重要。由于基序对自发转运没有简单的加性贡献，因此新型SMTP的合理工程设计仍然困难，这也为通过合成分子进化来寻找SMTP提供了更多理由。
