Weise Katrin, Triola Gemma, Janosch Sascha, Waldmann Herbert, Winter Roland
Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry, TU Dortmund University, Dortmund, Germany.
Biochim Biophys Acta. 2010 Jul;1798(7):1409-17. doi: 10.1016/j.bbamem.2009.12.006. Epub 2009 Dec 16.
In a combined chemical biological and biophysical approach, we studied the partitioning of differently fluorescent-labeled palmitoyl and/or farnesyl lipidated peptides, which represent membrane recognition model systems, as well as the full lipidated N-Ras protein into various model membrane systems including canonical model raft mixtures. To this end, two-photon fluorescence microscopy on giant unilamellar vesicles, complemented by tapping-mode atomic force microscopy (AFM) measurements, was carried out. The measurements were performed over a wide temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (solid-ordered (gel), fluid/gel, liquid-ordered/liquid-disordered, all-fluid). The results provide direct evidence that partitioning of the lipidated peptides and N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is liquid-disordered>liquid-ordered>>solid-ordered. Intriguingly, we detect - using the better spatial resolution of AFM - also a large proportion of the lipidated protein located at the liquid-disordered/liquid-ordered phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of neighboring domains. In an all-liquid-ordered, cholesterol-rich phase, phase separation can be induced by an effective lipid sorting mechanism owing to the high affinity of the lipidated peptides and proteins to a fluid-like lipid environment. At low temperatures, where the overall acyl chain order parameter of the lipid bilayer has markedly increased, such an efficient lipid sorting mechanism is energetically too costly and self-association of the peptide into small clusters takes place. These data reveal the interesting ability of the lipidated peptides and proteins to induce formation of fluid microdomains at physiologically relevant high cholesterol concentrations. Furthermore, our results reveal self-association of the N-Ras protein at the domain boundaries which may serve as an important vehicle for association processes and nanoclustering, which has also been observed in in vivo studies.
采用化学、生物学和生物物理学相结合的方法，我们研究了不同荧光标记的棕榈酰化和/或法尼基化脂质肽（代表膜识别模型系统）以及完全脂质化的N-Ras蛋白在各种模型膜系统中的分配情况，这些模型膜系统包括典型的模型筏混合物。为此，我们在巨型单层囊泡上进行了双光子荧光显微镜检查，并辅以轻敲模式原子力显微镜（AFM）测量。测量在较宽的温度范围内进行，范围从30到80摄氏度，以涵盖不同的脂质相态（固态有序（凝胶态）、流体/凝胶态、液态有序/液态无序、全流体态）。结果提供了直接证据，表明脂质化肽和N-Ras的分配优先发生在液态无序脂质域中，这也反映在更快的掺入动力学上。N-Ras与混合域脂质囊泡优先结合的相序为液态无序>液态有序>>固态有序。有趣的是，利用AFM更好的空间分辨率，我们还检测到很大一部分脂质化蛋白位于液态无序/液态有序相边界处，从而导致与相邻域边缘相关的线张力有利降低。在全液态有序、富含胆固醇的相中，由于脂质化肽和蛋白对类流体脂质环境的高亲和力，通过有效的脂质分选机制可以诱导相分离。在低温下，脂质双层的整体酰基链序参数显著增加，这种有效的脂质分选机制在能量上成本过高，肽会自组装成小簇。这些数据揭示了脂质化肽和蛋白在生理相关的高胆固醇浓度下诱导形成流体微域的有趣能力。此外，我们的结果揭示了N-Ras蛋白在域边界处的自组装，这可能是关联过程和纳米簇集的重要载体，这在体内研究中也已观察到。
