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。假设底物蛋白诱导的伴侣蛋白GroEL中的变构转变随结合蛋白底物的大小、结合亲和力、结合方向而变化，最终影响伴侣蛋白的功能和机制。利用低温电子显微镜的单粒子分析，在中等分辨率（13埃）下确定了七倍对称的groel底物蛋白复合物的结构。此外，从正常模式柔性拟合分析中确定的groel -底物蛋白复合物中发生的重要变构分子变化已经被定义。为了了解底物对伴侣蛋白影响机制的可变性，将使用单颗粒低温电镜获得各种GroEL底物蛋白复合物+ ADP的不对称结构。在底物蛋白结合过程中，GroEL中观察到的大量构象变化表明GroEL的变构转变不是两态的。对低分辨率多重变构结构态的解析，将使GroEL变构的结构图景更加全面。伴侣蛋白系统所采用的特定分类程序应该使其他人能够采用类似的方法来解决多个异质蛋白群体。更广泛的影响：使用低温电子显微镜进行单粒子分析的项目将有助于促进堪萨斯州内部的新合作，并将加强当地研究生和本科生的教育和培训（例如在斯克里普斯研究所的部分培训）。项目负责人、威廉·朱厄尔学院的斯科特·福尔克博士、堪萨斯大学的爱德华·果戈理和堪萨斯大学爱德华兹校区的大卫·普伦德格拉斯为本科生提供了研究伴侣蛋白制备、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.