Project 3. Chaperonin-mediated Assembly of alpha-Ketoacid Dehydrogenase Complexes


Recent studies have established that proper folding of some proteins depends on the presence of the protein machinery termed "molecular chaperones. Many members of this group are heat-shock proteins (Hsp's) that are highly conserved from bacteria to plants and humans. These molecular machines function by interacting with nascent or unfolded polypeptides to prevent misfolding, which would otherwise lead to aggregation. Defective protein folding has been implicated as a cause for human diseases such as cystic fibrosis, Alzheimer's disease and Huntington's disease.

Human mitochondrial chaperonins Hsp60/Hsp10 and their bacterial counterparts GroEL/GroES share 51% and 35% amino acid sequence identity, respectively. To date, the mode of action for chaperonin-mediated protein folding has been studied, in most part, using GroEL/GroES as a model system. Only limited information is available regarding the mechanism by which mitochondrial Hsp60/Hsp10 assist in protein folding and assembly. GroEL is a double-ring complex, and accumulated data have established that both cavities alternate in mediating protein folding, hence the term "two-stroke" engine. In contrast, human Hsp60 exists essentially in a single-ring to double-ring equilibrium. The difference in oligomeric assembly between GroEL and Hsp60 supports the notion that their reaction mechanisms are not necessarily identical.

Our laboratory has provided direct evidence that GroEL/GroES interact with a kinetically trapped heterodimeric assembly intermediate (model) during the folding and assembly of human E1b. GroEL/GroES promote dissociation/ reassociation cycles of the heterodimeric intermediate to facilitate its escape from the kinetic trap, resulting in productive alpha2beta2 heterotetrameric assembly. We hypothesize that a similar, but not identical, mechanism is utilized by Hsp60/Hsp10 to mediate efficient oligomeric assembly. In collaboration with Drs. Wah Chiu and Steve Ludtke of the National Center for Macromolecular Imaging at Baylor College of Medicine, we have recently achieved a reconstruction of native GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at 6- resolution. This high-resolution cryo-EM structure most likely reflects the true average solution conformation (GroEL cryo-EM structure). Based on these recent advances, our Specific Aims are as follows:

  1. To further elucidate the mode of interactions between chaperonins GroEL/GroES and assembly intermediates during oligomeric protein assembly in vitro

  2. To decipher the mechanism by which mitochondrial chaperonins Hsp60/Hsp10 promote folding and assembly of oligomeric proteins in vitro

  3. To investigate the role of chaperonins Hsp60/Hsp10 in mediating protein folding and assembly in vivo using the mitochondrial import system

Supported by the Welch Foundation grant I-1286