CAREER: Molecular Assemblies of Multidomain Signaling Protein on Membrane Surface

许多执行关键生命过程的蛋白质在结构上是模块化的，即由多个独立的结构域组成，这些结构域之间的分子内和分子间相互作用决定了功能。通常，导致激活的这些结构组织的变化发生在细胞膜上，实验上仍然无法接触到细胞膜。该项目将开发一种技术，以布鲁顿酪氨酸激酶(BTK)为模型系统，阐明膜表面的分子相互作用。该项目将开发实验工具，以阐明膜上生物分子组装的结构细节，以及对BTK和其他类似多域蛋白质的理解。与这一研究战略相结合，该教育计划寻求与科罗拉多州立大学SPUR校园合作，向公众灌输显微镜和单分子成像的概念。该计划基于这样的理念，即对科学图像的审美欣赏可以促进对科学的理解和亲和力。将自然概念结合在一起的艺术活动将被纳入到用艺术技术说明概念的创造性活动中。布鲁顿酪氨酸激酶(BTK)是一种在天然免疫和获得性免疫中都具有关键信号蛋白的多结构域蛋白，它在静止时保持自抑制状态，但在膜重新募集时变得活跃，从而经历戏剧性的结构变化。然而，很难研究膜的结构变化和分子相互作用，如齐聚或聚集，这些可能在细胞信号转导的功能结果中发挥关键作用。在本项目中，合成膜重构将与定量光谱方法相结合，系统地探索BTK膜特有的超分子组装，具体目标如下：(1)确定全长BTK在膜上的多聚状态，以及导致多聚的特定分子相互作用，(2)评估分子组装对BTK活化和催化活性的影响，以及(3)确定宏观BTK脂结合结构域簇的物质状态。总体而言，该项目将促进我们在细胞信号方面的知识，特别是那些具有特定于细胞膜的紧急特性的知识。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Many proteins that carry out critical life processes are modular in structure, i.e., composed of multiple independent domains, and the intra- and intermolecular interactions among these domains determines function. Often, changes in these structural organization that leads to activation occurs at the cellular membrane, which remains experimentally inaccessible. This project will develop a technology that can elucidate the molecular interactions on membrane surfaces using Bruton’s tyrosine kinase (Btk) as a model system. The project will develop experimental tools for elucidating structural details for biomolecular assemblies on membranes, as well as the understanding of Btk and other similar multidomain proteins. Integrated with this research strategy, the education plan seeks to instill the concepts of microscopy and single-molecule imaging in the public, in collaboration with the Colorado State University Spur Campus. The plan is based on the idea that aesthetic appreciation for scientific imageries can facilitate the understanding and affinity towards science. Artistic actives that combine natural concepts will be incorporated into creative activities in which artistic techniques illustrate the concepts.Bruton’s tyrosine kinase (Btk), a key signaling protein in both innate and adaptive immunity, is a multidomain protein that maintains an autoinhibited state at rest but undergoes a dramatic structural change upon membrane recruitment where it becomes active. However, it is very difficult to study membrane-specific structural changes and molecular interaction such as oligomerization or clustering that can play a critical role in the functional outcome of cellular signaling. In this project, synthetic membrane reconstitution will be combined with quantitative spectroscopic methods to systematically probe the membrane-specific supramolecular assemblies of Btk, with the following specific objectives: (1) determine the multimerization state of the full-length Btk on membranes, and the specific molecular interactions responsible for the multimerization, (2) evaluate the molecular assembly’s impact on Btk’s activation and catalytic activity, and (3) identify the material state of the macroscopic Btk lipid binding domain clusters. Overall, the project will advance our knowledge in cellular signaling, particularly those with emergent properties specific to cellular membranes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.