Influence of the Lipid Anchor Motif of N-Ras on the Interaction with Lipid Membranes: A Surface Plasmon Resonance Study
Influence of the Lipid Anchor Motif of N-Ras on the Interaction with Lipid Membranes: A Surface Plasmon Resonance Study
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DOI：
10.1016/j.bpj.2010.02.005
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发表时间：
2010-05-19
影响因子：
3.4
通讯作者：
Winter, Roland
中科院分区：
文献类型：
--
作者：
Gohlke, Andrea;Triola, Gemma;Winter, Roland
GTP酶是否在参与细胞分化和增殖的信号转导通路中发挥关键作用，而膜结合是其正常功能所必需的。为了确定脂锚基序的性质以及N-RAS的活性形式(GTP)和非活性形式(GDP)之间的差异对脂膜上的分配和定位的影响，合成了5种不同的N-RAS结构(Far/Far(GDP)、HD/Far(GDP)、HD/HD(GDP)、Hd/HD(GDP)、Far(GDP)和HD/Far(GppNHp))。利用表面等离子体共振技术，我们能够跟踪脂化蛋白质进入和离开模型膜的过程，模型膜由纯液体有序(I-O)或液体无序(I-D)相和非均相两相混合物组成，即I-O+I-D相共存的RAFT混合物。此外，我们还考察了带负电荷的头基和储存的曲率弹性应力对脂化N-RAS蛋白结合性质的影响。在大多数情况下，不同的锚定基序存在显著差异。一般来说，N-RAS蛋白优先插入到流体状的，而不是刚性的，有序的脂质双层环境中。与脂头基团的静电相互作用或膜的储存曲率弹性应力似乎对脂化蛋白质的结合和解离过程没有太大影响。在类流体膜中，单芳基N-RAS的缔合率最高，解离速度最快。蛋白质的双脂化，特别是包括法尼化，导致初始结合率显著降低，但最终结合强烈。天然N-RAS HD/FAR的核苷酸负载量的变化导致了略有不同的结合和解离动力学，以及结合的稳定性，似乎影响了膜平面横向分离的倾向。与GDP结合的非活性形式的N-RAS与HD/FAR锚点显示出更强的膜结合，这可能是由于与活性GTP结合的形式相比，更明显的在膜基质中自组装的倾向。
Has GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (I-o) or liquid-disordered (I-d) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with I-o + I-d phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.