Structural and Energetic Effects of Protein Substrate Binding on GroEL Chaperonin

该项目的长期目标是确定底物特异性如何影响伴侣蛋白的机制、结构和变构转变+ADP。假设底物蛋白诱导伴侣蛋白GroEL中的变构转变随结合蛋白底物的大小、结合亲和力、结合方向而不同，最终影响伴侣蛋白的功能和机制。利用冷冻电子显微镜的单颗粒分析，在中等分辨率(13埃)下确定了一个七重对称化的GroEL底物蛋白质复合体的结构。此外，还定义了由简正模式柔性拟合分析确定的GroEL-底物蛋白质复合体中发生的重要变构分子变化。为了了解底物的变化对伴侣作用机制的影响，我们将用单颗粒的冷冻电子显微镜获得不同GroEL底物蛋白质复合体+ADP的不对称结构。在底物蛋白结合过程中观察到GroEL的大量构象变化，表明GroEL变构转变不是两态的。解决低分辨率的多重变构结构状态将导致更全面的GroEL变构结构图景。用于伴侣系统的特定分类程序应使其他人能够使用类似的方法来解析多个不同的蛋白质种群。广泛的影响：使用单粒子分析和低温电子微粒子的项目的存在将有助于促进堪萨斯州内部的新合作，并将加强对当地研究生和本科生的教育和培训(例如，斯克里普斯研究所的部分培训)。威廉·珠尔学院的Scott Falke博士、UMKC的Edward Gogol博士和KU Edwards校区的David Prendergras为本科生提供了从事伴侣制备、电子显微镜图像收集和图像分析等各种生物物理方面的工作的机会。

The long-term goal of this project is to determine how substrate specificity influences chaperonin mechanism, structure and allosteric transition + ADP. It is hypothesized that the substrate protein induced allosteric transitions in the chaperonin GroEL vary with size, binding affinity, binding orientation of the bound protein substrate, ultimately influencing chaperonin function and mechanism. Using single particle analysis using cryo-electron microscopy, the structure of a seven-fold symmetrized GroELsubstrate protein complex at an intermediate resolution (13 angstroms) has been determined. In addition, the important allosteric molecular changes that occur in GroEL-substrate protein complexes as determined from normal mode flexible fitting analysis have been defined. To understand the variability of substrate influences on the chaperonin mechanism, unsymmetrized structures of various GroEL substrate protein complexes + ADP will be obtained using cryo-electron microscopy of single particles. The observed presence of a multitude of conformational changes in GroEL during substrate protein binding suggests that GroEL allosteric transitions are not two-state. Resolving the low resolution multiple allosteric structural states will lead to a more comprehensive structural picture of GroEL allostery. The specific classification procedures employed for the chaperonin system should enable others to employ similar methods to resolve multiple heterogeneous protein populations.Broader impacts: The presence of projects using single particle analysis with cryoelectron microscipy will help foster new collaborations within Kansas and will enhance the education and training of local graduate students and undergraduate students (e.g. partial training at the Scripps Research Institute). The PI, Dr. Scott Falke at William Jewel College, Edward Gogol at UMKC and David Prendergrass at the KU Edwards campus have set up opportunities for undergraduates to work on various biophysical aspects of chaperonin preparation, electron microscopy image collection and image analysis.