Laboratório de Biofísica Molecular, Departamento de Física, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Grupo de Biofísica Molecular "Sérgio Mascarenhas", Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
Biochim Biophys Acta Gen Subj. 2018 Apr;1862(4):855-865. doi: 10.1016/j.bbagen.2018.01.009. Epub 2018 Jan 13.
Golgi Reassembly and Stacking Proteins (GRASPs) are widely spread among eukaryotic cells (except plants) and are considered as key components in both the stacking of the Golgi cisternae and its lateral connection. Furthermore, GRASPs were also proved essential in the unconventional secretion pathway of several proteins, even though the mechanism remains obscure. It was previously observed that the GRASP homologue in Cryptococcus neoformans has a molten globule-like behavior in solution.
We used circular dichroism, synchrotron radiation circular dichroism and steady-state as well as time-resolved fluorescence.
We report the disorder-to-order transition propensities for a native molten globule-like protein in the presence of different mimetics of cell conditions. Changes in the dielectric constant (such as those experienced close to the membrane surface) seem to be the major factor in inducing multiple disorder-to-order transitions in GRASP, which shows very distinct behavior when in conditions that mimic the vicinity of the membrane surface as compared to those found when free in solution. Other folding factors such as molecular crowding, counter ions, pH and phosphorylation exhibit lower or no effect on GRASP secondary structure and/or stability.
To the best of our knowledge, this is the first study focusing on understanding the disorder-to-order transitions of a molten globule structure without the need of any mild denaturing condition. A model is also introduced aiming at describing how the cell could manipulate the GRASP sensitivity to changes in the dielectric constant during different cell-cycle periods.
高尔基重组和堆积蛋白（GRASPs）广泛存在于真核细胞中（植物除外），被认为是高尔基体潴泡堆叠及其侧向连接的关键组成部分。此外，GRASPs 还被证明是几种蛋白质非常规分泌途径所必需的，尽管其机制尚不清楚。先前观察到，新生隐球菌中的 GRASP 同源物在溶液中具有类无规卷曲的行为。
我们使用了圆二色性、同步辐射圆二色性以及稳态和时间分辨荧光。
我们报告了在不同细胞条件模拟物存在下，天然类无规卷曲蛋白的无序到有序转变趋势。介电常数的变化（如在接近膜表面时经历的变化）似乎是诱导 GRASP 发生多次无序到有序转变的主要因素，与在溶液中自由状态相比，GRASP 在模拟接近膜表面的条件下表现出非常不同的行为。其他折叠因素，如分子拥挤、抗衡离子、pH 值和磷酸化，对 GRASP 的二级结构和/或稳定性的影响较低或没有。
据我们所知，这是第一项研究，旨在在无需任何温和变性条件的情况下，深入了解无规卷曲结构的无序到有序转变。还引入了一个模型，旨在描述细胞如何在不同的细胞周期中操纵 GRASP 对介电常数变化的敏感性。
