MICROTUBULE MOTORS STUDIED ON A MOLECULAR SCALE

分子尺度上的微管电机研究

• 批准号: 2189990
• 负责人: STEVEN M BLOCK
• 金额: $ 20.1万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2189990
• 关键词: adenylate kinase; biomedical equipment development; chemical models; computer program /software; interferometry; kinesin; microtubule associated protein; microtubules; model design /development; protein structure function; recombinant proteins; structural biology

Motor proteins, or mechanoenzymes, convert metabolic energy into force, generating movement in all living organisms. The largest class of such proteins derives energy from the hydrolysis of ATP, and includes the myosin, dynein, and kinesin superfamilies. Despite over a century of study and the arsenal of biochemical and biophysical approaches that have been tried, the molecular basis by which motor proteins work remains obscure. Today, the mechanism of force production by proteins is one of the great outstanding problems in biology, with obvious implications in understanding the basis of motor-related disease. The advent of in vitro motility assays has, at last, allowed mechanoenzymes to be studied in comparative isolation, using purified components interacting in defined experimental geometries. Such systems hold great promise because they facilitate genetic, biochemical, physical and molecular biological manipulations not possible in complex cellular systems. Recent advances, described here, show that physiology is feasible at the level of individual molecules, in an assay using kinesin motors moving along microtubules. The kinesin-microtubule system is particularly amenable to study, because (1) movement is produced by single motors, (2) motion is slow enough for measurement, (3) microtubules can be seen in the light microscope, (4) both recombinant kinesin (and kinesin-like) proteins and protein fragments, expressed in either bacterial or eukaryotic vectors, move in vitro, and (5) methods now exist to produce controlled forces and measure displacements on a molecular scale. The long-term goal of this research is to develop a molecular model for motor protein function, based on detailed physical knowledge combined with biochemical/biostructural information. Specific aims include measurement of the speeds, forces, displacements, cycle timing, ATP coupling, and other properties of native kinesin, recombinant kinesin (and engineered constructs), and the minus end-directed motor ncd. For this purpose, advanced instrumentation based on optical trapping ('optical tweezers') and optical interferometry will be built and used to track motion at the subnanometer level. Closely-related apparatus will also be used to compile data on the micromechanical properties of kinesin/microtubules, and to accomplish a new kind of imaging, 'optical force microscopy,' that may provide insights into the molecular structures that underlie motility.

运动蛋白或机械酶，将代谢能力转化为 在所有生物体中产生运动。 最大的阶级 蛋白质从ATP的水解中得出能量，包括 肌球蛋白，动力蛋白和动力素超家族。 尽管一个多世纪 研究和具有生化和生物物理方法的武器库 尝试了运动蛋白的作用的分子基础 朦胧。 如今，蛋白质产生力的机理是一种 生物学上的巨大问题，在 了解与运动相关疾病的基础。 体外的出现 运动测定最终允许将机械酶研究 比较隔离，使用定义的纯化组件相互作用 实验几何形状。 这样的系统具有巨大的希望，因为他们 促进遗传，生化，物理和分子生物学 在复杂的细胞系统中不可能进行操作。 最近的进步 在此描述，证明生理学在 单个分子，在使用驱动蛋白电动机的测定中 微管。 驱动蛋白 - 微管系统特别适合 研究，因为（1）移动是由单电机产生的，（2）运动 足够慢以进行测量，（3）在光线下可以看到微管 显微镜，（4）重组驱动蛋白（和驱动蛋白样）蛋白和 蛋白质片段，在细菌或真核载体中表达， 在体外移动，现在存在（5）方法来产生受控力和 测量分子尺度的位移。 这个长期目标 研究是为运动蛋白功能开发分子模型， 基于详细的物理知识与 生化/生物结构信息。 具体目的包括测量 速度，力，位移，周期时机，ATP耦合和 本地驱动蛋白，重组驱动蛋白的其他特性（并设计 构造）和减去端导向的电动机NCD。 为此， 基于光学诱捕的高级仪器（“光学镊子”） 和光学干涉法将被建造并用于跟踪运动 亚纳光级。 密切相关的设备也将用于 有关驱动蛋白/微管的微力特性的编译数据， 并完成一种新型的成像，即“光学显微镜”， 可以提供有关基于的分子结构的见解 运动。