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

职业：综合生物合成和单分子方法研究集体运动蛋白动力学

• 批准号: 0643832
• 负责人: Michael Diehl
• 金额: $ 54.9万
• 依托单位: William Marsh Rice University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0643832&HistoricalAwards=false
• 关键词: CAREER; Integrated; Biosynthetic; Single; Molecule

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.

运动蛋白之间的集体相互作用在细胞内货物的运输中起着至关重要的作用，它提供了一种优化细胞质异质环境传输的方法。 结果，集体生物运动转运对于维持健康的细胞功能至关重要。但是，仍然存在重大挑战，从根本上了解运动蛋白如何集体发挥作用。 这些挑战在很大程度上是源于连接结构信息的实验困难，该结构信息描述了运动蛋白相互作用组的超分子特性与细胞内转运的集体模式。 这项研究将通过建立能够从结构和动力学的角度研究集体运动运输的技术来弥合这些差距。 该目标将通过开发可用于构建相互作用运动蛋白的实验模型系统的生物合成技术来实现。 基于自组装的DNA纳米结构和工程人工蛋白，这些技术将被利用以构建分子平台，以使组件中包含的位置，数量和类型的电动机以及运动互连的弹性在分子水平上被编码。 将使用基于总内部反射显微镜（TIRFM）的新型单分子成像方法来表征组件的超分子结构。 此外，将使用光学诱捕仪器研究集体运动动力学。 该设备将允许通过单分子分辨率和实时研究相互作用的运动蛋白的复杂踏脚力学。 总之，这两种技术都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，负载依赖性，步长，停留时间等）如何取决于组件的分子结构。 由于已知生物机构的机械化学强烈取决于应变，因此基于电动机之间的机械耦合，因此建立这些关系将洞悉电动机之间的共享力如何影响其机械化学化学。 此外，通过开发一种平台技术来构建和探测各种富含建筑的运动蛋白系统，这项研究将允许集体运输机制通常被蜂窝环境的复杂性掩盖，以精确确定。 这项研究的哲学框架还在一项教育计划中利用，该计划旨在通过使他们拥有在生物科学和生物工程中面对边境挑战所必需的知识基础和技能来增强学生和教育工作者的科学素养。 这些目标将通过使用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，这项工作纳入了旨在使高中生接受大学级别的研究和教育的外展活动。 这些活动还将有助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者传播新的教学工具。