Vagt Toni, Zschörnig Olaf, Huster Daniel, Koksch Beate
Institute of Chemistry and Biochemistry-Organic Chemistry, Free University of Berlin, Takustrasse 3, 14195 Berlin, Germany.
Chemphyschem. 2006 Jun 12;7(6):1361-71. doi: 10.1002/cphc.200600010.
We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.
我们引入了一种全新设计的肽模型系统，该系统能够对以下内容进行系统研究：1）膜环境在卷曲螺旋肽折叠中的作用；2）α-螺旋卷曲螺旋七肽重复序列的不同结构域对与膜相互作用的影响；3）卷曲螺旋肽与膜相互作用的动力学如何依赖环境条件。从理想的α-螺旋卷曲螺旋肽序列出发，设计了几种带正电荷的类似物，它们对带负电荷的脂质膜具有很高的亲和倾向。此外，这些肽形成稳定α-螺旋卷曲螺旋结构的能力各不相同。研究了膜环境对肽折叠的影响。所有带正电荷的肽都与带负电荷的膜表现出强烈的相互作用。如圆二色性测量所示，这种相互作用诱导了先前无规卷曲肽的α-螺旋结构。此外，囊泡结合肽的卷曲螺旋相互作用诱导了囊泡聚集。动态光散射实验表明，囊泡聚集的强度随着肽形成稳定α-螺旋卷曲螺旋的内在能力而增加。因此，具有最强螺旋间和螺旋内卷曲螺旋相互作用的肽变体对囊泡聚集的影响最强。研究了该肽在膜结合状态下的二级结构及其对磷脂的影响。通过（13）C交叉极化魔角旋转核磁共振实验分析了肽-脂质聚集体内的肽构象。在肽链的第12位引入了一个均匀（13）C和（15）N标记的亮氨酸残基。（13）C化学位移和扭转角测量结果支持了该肽在膜结合状态下为α-螺旋结构的发现。肽结合后未观察到膜泄漏或融合现象，这对于两亲性肽结构来说是不寻常的。我们的结果为在分子水平上系统研究α-螺旋卷曲螺旋折叠基序在膜活性事件中的影响奠定了基础。
