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.
