GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor and, when bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the protein's secondary structure, according to our circular dichroism data, it had a significant impact on the protein's thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

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肉豆蔻酰化及其对人高尔基体重组和堆积蛋白 55 的影响

GRASP55 是一种豆蔻酰化蛋白，位于内侧/跨高尔基体面部，参与高尔基体结构维持和非常规分泌途径的调节。据信 GRASP55 通过充当束缚因子在高尔基体中实现其主要功能，并且当与脂质双层结合时，其相对于膜表面的方向受到限制，以确定其正确的反式寡聚化。尽管肉豆蔻酰化在 GRASP 功能中起着至关重要的作用，但这种蛋白质修饰对膜锚定特性和 GRASP 结构组织的影响仍然难以捉摸。在这里，介绍了 GRASP55 膜锚定域的大肠杆菌中豆蔻酰化的优化方案。肉豆蔻酰化/非肉豆蔻酰化 GRASP55 GRASP 结构域的生物物理特性在膜模拟胶束环境中表征。尽管肉豆蔻酰化不会对蛋白质的二级结构造成任何影响，但根据我们的圆二色性数据，它对蛋白质的热稳定性和溶解性产生了重大影响。与两种 GRASP55 构建体一起孵育的带负电荷的脂质体的电泳仅对肉豆蔻酰化锚定蛋白显示出不同的电泳迁移率，从而证明肉豆蔻酰化对于生物膜锚定是必不可少的。分子动力学模拟用于进一步探索确定膜中 GRASP 受限方向的锚定过程。尽管肉豆蔻酰化不会对蛋白质的二级结构造成任何影响，但根据我们的圆二色性数据，它对蛋白质的热稳定性和溶解性产生了重大影响。与两种 GRASP55 构建体一起孵育的带负电荷的脂质体的电泳仅对肉豆蔻酰化锚定蛋白显示出不同的电泳迁移率，从而证明肉豆蔻酰化对于生物膜锚定是必不可少的。分子动力学模拟用于进一步探索确定膜中 GRASP 受限方向的锚定过程。尽管肉豆蔻酰化不会对蛋白质的二级结构造成任何影响，但根据我们的圆二色性数据，它对蛋白质的热稳定性和溶解性产生了重大影响。与两种 GRASP55 构建体一起孵育的带负电荷的脂质体的电泳仅对肉豆蔻酰化锚定蛋白显示出不同的电泳迁移率，从而证明肉豆蔻酰化对于生物膜锚定是必不可少的。分子动力学模拟用于进一步探索确定膜中 GRASP 受限方向的锚定过程。与两种 GRASP55 构建体一起孵育的带负电荷的脂质体的电泳仅对肉豆蔻酰化锚定蛋白显示出不同的电泳迁移率，从而证明肉豆蔻酰化对于生物膜锚定是必不可少的。分子动力学模拟用于进一步探索确定膜中 GRASP 受限方向的锚定过程。与两种 GRASP55 构建体一起孵育的带负电荷的脂质体的电泳仅对肉豆蔻酰化锚定蛋白显示出不同的电泳迁移率，从而证明肉豆蔻酰化对于生物膜锚定是必不可少的。分子动力学模拟用于进一步探索确定膜中 GRASP 受限方向的锚定过程。