Dynamics and equilibria of weak in-cell interactions

为了生存，细胞必须传递信号，比如“攻击这个病毒！”或者构建微小的结构，比如剪接体，剪接体是一种微小的粒子，可以剪切和剪接遗传密码，使其准备好制造蛋白质。所有这些都涉及到蛋白质和核酸在细胞内的折叠和组装。在过去，这主要是在试管中测量的，但它实际上发生在细胞内。该项目将观察单个细胞内部，以准确测量细胞和试管之间折叠和结合的差异。除了向我们展示这些分子水平的事件在现实生活中如何在人类细胞内发生外，该项目还将培养分子生物物理学现代技术方面的几名博士生和一名博士后。它还支持对本科生研究人员的培训，使他们早在博士阶段之前就能获得实践研究技能。实验室成员将参加科学多样性的拓展和会议，以帮助研究企业反映美国公民的多样性。提出的研究目标，重点是活细胞和生物体内部蛋白质和RNA的相互作用，将通过结合新的微扰显微镜技术（细胞周围的温度、渗透压和溶剂条件突然跳跃）和细胞内部的大规模原子模拟（使用能够在数百微秒内模拟数百万个原子的超级计算机）来实现。要研究的关键系统都围绕着生物分子-生物分子组装（结合）和信号传导。微管蛋白二聚体（细胞骨架的一部分）的组装将在细胞内成像，使用一种特殊的微管蛋白变体，这种变体不会破坏健康细胞中其他微管蛋白的活性。将在活细胞内研究数百种衣壳蛋白的病毒衣壳组装。当病毒感染时，这种组装反应是如何向后移动的，而当病毒复制并准备离开细胞时，这种组装反应是如何向前移动的？U1A剪接体颗粒切割前信使RNA以制造mRNA，其组装将在细胞核内外的复杂周期中受到监测。将在细胞内和试管中比较热休克伴侣与客户和辅助因子的组装，以避免蛋白质的错误折叠。活体脊椎动物体内双结构域蛋白的折叠将首次被成像。不同的组织如何以不同的方式促进蛋白质折叠？微秒级的全细胞反应扩散实验可以与全细胞模型进行比较，也可以与部分细胞质的分子动力学模拟进行比较。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

In order to survive, cells must transmit signals, such as "attack this virus!" or build tiny structures, such as the spliceosome, a microscopic particle that cuts and splices the genetic code to get it ready to make proteins. All this involves proteins and nucleic acids folding and assembling inside the cell. In the past, this has been measured primarily in test tubes, but it really happens inside cells. This project will look inside single cells to measure accurately just how much folding and binding differ between cell and test tube. In addition to showing us how these molecular-level events happen in real life inside human cells, the project will train several PhD students and a postdoctoral fellow in modern techniques of molecular biophysics. It also supports training of undergraduate researchers to gain hands-on research skills long before the PhD stage. Lab members will participate in diversity-in-science outreach and meetings, to help the research enterprise reflect the diversity of US citizens.The proposed research goals, focusing on protein and RNA interactions inside living cells and organisms, will be achieved by combining new perturbation microscopy techniques (where temperature, osmotic pressure, and solvent conditions around cells are suddenly jumped) with large scale atomistic simulations of the interior of the cell (using supercomputers capable of simulating millions of atoms over hundreds of microseconds). The key systems to be investigated all revolve around biomolecule-biomolecule assembly (binding) and signaling. Assembly of tubulin protein dimers (part of the cell's skeleton) will be imaged inside cells, using a special tubulin variant that does not disrupt other tubulin activity in healthy cells. Assembly of viral capsids from hundreds of capsid proteins will be studied inside live cells. How does this assembly reaction manage to move backward when a virus infects, but forward when it is replicated and ready to leave the cell? Assembly of the U1A spliceosomal particle, which cuts pre-messenger RNA to make mRNA, will be monitored in its complex cycle both inside and outside of the nucleus. Assembly of heat shock chaperones with their clients and auxiliary factors to save proteins from misfolding will be compared in-cell and in the test tube. Folding of a two-domain protein inside a living vertebrate organism will be imaged for the first time. How do different tissues promote protein folding differently? Microsecond reaction-diffusion experiments in the whole cell at once will enable comparison with whole-cell models, as well as with molecular dynamics simulations of parts of the cytoplasm.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.