Dynamic Interactions between Intrinsically Disordered Proteins and Curved Membrane Surfaces

项目摘要/摘要。固有无序蛋白(IDPs)约占人类的40% 蛋白质组，并与许多人类疾病有关，包括神经疾病和癌症。 因此，在过去十年中，国内流离失所者的生物学相关性引起了人们的极大兴趣。国内流离失所者经常 与弯曲的膜相互作用，形成细胞生理所必需的结构，如突触和 内吞小泡。这些相互作用是由膜曲率传感促进的。少年派最近发现 IDPs是膜曲率的有效传感器。这一发现实质上背离了 主要的结构-功能范式，因为国内流离失所者缺乏固定的三维结构，而且往往是错误的 推测也缺乏生物物理功能。国内流离失所者的曲率敏感性，以及许多其他类型的 蛋白质，通常是在热力学平衡条件下研究的。然而，膜相互作用的蛋白质存在于 它们的膜结合状态和膜非结合状态之间的动态平衡。可能需要几分钟的时间 实现蛋白质和膜之间的热力学平衡，但细胞过程，如细胞 信令，发生在毫秒到秒之间的任何地方。从时间尺度上的这种不匹配可以清楚地看到 当仅考虑热力学平衡时，与时间尺度有关的动态信息 细胞过程被省略了。很少或根本没有文献研究这种现象，可能是因为 与实现这种实验测量相关的困难。因此，动态交互作用在 蛋白质和弯曲的膜结构知之甚少。利用他们在量化方面的专业知识 荧光显微镜和蛋白质工程，PI实验室的目标是开发和应用 允许直接可视化和描述动态交互的技术和策略 在国内流离失所者和弯曲的膜结构之间。国际和平研究所未来的研究计划包括3个主要方面 研究项目。项目1中的工作将评估蛋白质结构影响吸附和 解吸动力学，测试弯曲膜影响各种蛋白质结构的工作假说 不同的。项目2将评估蛋白质网络对国内流离失所者结合动态的影响， 回答有关蛋白质多价态对动态行为的影响的问题。在项目3中工作将 开发模拟细胞内环境的实验技术和策略，回答问题 关于IDPs与弯曲的膜基质之间的动态相互作用，在两种- 膜表面的三维、相分离的蛋白质混合物或三维蛋白质 散装解决方案中的聚集体。正如前面提到的，我们目前对相互作用的理解是 蛋白质和弯曲的膜来自测量蛋白质分配的体系。 在达到热力学平衡后。相比之下，拟议的工作将填补现有工作的一个关键空白 通过直接观察和量化蛋白质和弯曲的膜之间的动态相互作用来获得知识。

Project Summary/Abstract. Intrinsically Disordered Proteins (IDPs) represent approximately 40% of the human proteome and are implicated in a large number of human diseases, including neurological disorders and cancer. Therefore, the biological relevance of IDPs has garnered substantial interest over the last decade. IDPs often interact with curved membranes to form structures that are essential to cellular physiology, such as synaptic and endocytic vesicles. These interactions are facilitated by membrane curvature sensing. The PI recently discovered that IDPs are potent sensors of membrane curvature. This discovery is a substantive departure from the predominant structure-function paradigm, as IDPs lack fixed three-dimensional structure and are often incorrectly assumed to also lack biophysical functionality. The curvature sensitivity of IDPs, as well as many other types of proteins, is normally studied at thermodynamic equilibrium. However, membrane-interacting proteins exist in a dynamic equilibrium between their membrane-bound and membrane-unbound states. It can take minutes to achieve thermodynamic equilibrium between proteins and membranes, but cellular processes, such as cell signaling, occur anywhere between milliseconds to seconds. It is clear from this mismatch in timescales that when thermodynamic equilibrium alone is considered, dynamic information that is pertinent to the timescale of cellular processes is omitted. Little to no literature exists that examines this phenomenon, likely owing to the difficulty associated with achieving such experimental measurements. Thus, dynamic interactions between proteins and curved membrane structures are poorly understood. Using their expertise in quantitative fluorescence microscopy and protein engineering, the goal of the PI's laboratory is to develop and apply techniques and strategies that will allow for direct visualization and characterization of dynamic interactions between IDPs and curved membrane structures. The PI's future research program contains 3 overarching research projects. Work in Project 1 will evaluate the extent to which protein structure influences adsorption and desorption kinetics, testing the working hypothesis that curved membranes affect various protein structures differently. Work in Project 2 will evaluate the impact of protein networks on the binding dynamics of IDPs, answering questions about the influence of protein multivalency on dynamic behavior. Work in Project 3 will develop experimental techniques and strategies that mimic the intracellular environment, answering questions about dynamic interactions between IDPs and curved membrane substrates that occur in the presence of two- dimensional, phase separated protein mixtures on the membrane surface or three-dimensional protein aggregates in the bulk solution. As previously mentioned, our current understanding of the interactions between proteins and curved membranes was derived from systems in which the partitioning of proteins was measured after thermodynamic equilibrium was achieved. In contrast, the proposed work will fill a key gap in existing knowledge by directly observing and quantifying dynamic interactions between proteins and curved membranes.