CAREER: Integrated Biosynthetic and Single-Molecule Approaches to Investigate Collective Motor Protein Dynamics

马达蛋白之间的集体相互作用为细胞质的异质环境提供了一种优化运输的手段，在细胞内货物的运输中发挥着至关重要的作用。因此，集体生物运动运输对于维持健康的细胞功能是必不可少的。然而，从根本上理解马达蛋白的集体功能仍然是一个重大的挑战。这些挑战在很大程度上源于将描述相互作用的马达蛋白基团的超分子性质的结构信息与细胞内运输的集体模式联系起来的实验困难。这项研究将通过建立能够从结构和动力学角度研究集体汽车运输的技术来弥合这些差距。这一目标将通过开发可用于构建相互作用的马达蛋白的实验模型系统的生物合成技术来实现。基于自组装DNA纳米结构和工程人造蛋白质，这些技术将被利用来构建分子平台，使组装中包含的马达的位置、数量和类型以及马达互连的弹性能够在分子水平上进行编码。基于全内反射显微镜(TIRFM)的新型单分子成像方法将表征这些组件的超分子结构。此外，还将使用光学捕获仪器来研究集体马达的动力学。这一装置将允许以单分子分辨率和实时研究相互作用的马达蛋白质的复杂的步进机制。这两种技术结合在一起，将有助于开发详细的结构-活性关系，这些关系定义了关键的传输属性(速度、负载依赖、步长、停留时间等)取决于组件的分子结构。由于生物马达的机械力化学强烈依赖于应变，从而依赖于马达之间的机械耦合，因此建立这些关系将有助于深入了解马达之间的力分配如何影响它们的机械力化学。此外，通过开发一种平台技术来构建和探索各种结构丰富的马达蛋白系统，这项研究将使人们能够准确确定通常被复杂的细胞环境掩盖的集体运输机制。这项研究的哲学框架还被纳入一项教育计划，该计划努力提高学生和教育工作者的科学素养，使他们具备应对生物科学和生物工程领域前沿挑战所需的知识基础和技能。这些目标将通过将这项研究作为为研究生和本科生创造研究和教育机会的基础来实现。此外，这项工作还包括旨在让高中生接触大学水平的研究和教育的外联活动。这些活动还将用于招募代表性不足的少数群体进行生物科学研究，并将用于向高中教育工作者传播新的教学工具。

Collective interactions between motor proteins play an essential role in the transport of intracellular cargo by providing a means to optimize transport for the heterogeneous environment of the cytoplasm. As a result, collective biomotor transport is essential for the maintenance of healthy cellular function. However, significant challenges remain to fundamentally understand how motor proteins function collectively. These challenges largely stem from experimental difficulties in connecting structural information that describes the supramolecular properties of interacting groups of motor proteins to collective modes of intracellular transport. This research will bridge these gaps by establishing techniques that enable collective motor transport to be investigated from both a structural and dynamical standpoint. This goal will be accomplished by developing biosynthetic technologies that can be used to construct experimental model systems of interacting motor proteins. Based on self-assembled DNA nanostructures and engineered artificial proteins, these technologies will be harnessed to build molecular platforms that enable the position, number, and type of motors contained in an assembly, as well as the elasticity of motor interconnects to be encoded at the molecular level. The supramolecular architecture of the assemblies will be characterized using novel single-molecule imaging methods based on total-internal reflection microscopy (TIRFM). In addition, collective motor dynamics will be investigated using optical trapping instrumentation. This apparatus will allow the intricate stepping mechanics of interacting motor proteins to be investigated with single-molecule resolution and in real time. Together, both techniques will facilitate development of detailed structure-activity relationships that define how critical transport properties (velocities, load dependence, step size, dwell times, etc...) depend on the molecular architecture of assemblies. Because the mechano-chemistry of biomotors is known to be strongly dependent on strain, and hence on the mechanical coupling between motors, establishing these relationships will provide insight into how sharing force between motors influences their mechano-chemistry. Furthermore, by developing a platform technology to construct and probe a variety of architecturally rich systems of motor proteins, this research will allow mechanisms of collective transport that are typically masked by the complexities of cellular environments to be precisely determined. The philosophical framework of this research is also harnessed in an education plan that strives to enhance the scientific literacy of students and educators by empowering them with the knowledge base and skills necessary to confront frontier challenges in the biosciences and bioengineering. These goals will be accomplished by using this research as a foundation for creating research and education opportunities for both graduate and undergraduate students. Furthermore, this work incorporates outreach activities designed to expose high school students to college-level research and education. These activities will also serve to recruit under-represented minority groups to bioscience research and will be used to disseminate new instructional tools to high school educators.