Double-ring chaperonin complexes are essential mediators of cellular protein folding. Based on their ability to bind unfolded polypeptides within their ring cavities, they prevent off-pathway reactions and promote productive protein folding to the native state in an ATP-dependent manner. Partial folding appears in the chaperonin cavity before release of the substrate proteins into solution. The functional principles of the bacterial GroEL/GroES chaperonin system have been derived from in vitro reconstitution studies, the exact mechanism of its action is however unclear. Unlike the homo-oligomeric GroEL, the only recently discovered eukaryotic cytosolic chaperonin TRiC is a hetero-oligomeric complex and appears not to rely on a GroES-like cofactor. Little is known about the function and substrate specificity of this chaperonin. The goal of this proposal is to understand the molecular mechanism by which chaperonin ring complexes mediate cytosolic protein folding. The following specific aspects will be addressed with the purified components in vitro: Folding assays with immobilized chaperonin complexes and trap chaperonins will be used in experiments to determine the mechanism of GroEL/GroES and TRiC action. To analyze how ATP hydrolysis during the chaperonin reaction cycle is coupled to protein folding and release, the modified chaperonins with defects in this process will be examined. These studies will be done in parallel with the prokaryotic and eukaryotic chaperonins to learn whether they facilitate protein folding in a similar manner. The conformation of chaperonin-bound proteins will be assessed by ligand and inhibitor binding characteristics of substrate proteins by monitoring fluorescence changes of intrinsic and extrinsic substrate fluorophors. The substrate spectrum of TRiC, the kinetics of substrate folding and release will be analyzed, and two-domain fusion proteins will serve as model systems for the analysis of domain-wise folding. As a major step towards the goal of reconstituting cellular folding in vitro, the proposed biochemical assays will be performed also in the presence of macromolecular crowding agents and concentrated cytoplasmic protein extracts which closely mimic the cellular milieu and affect profoundly the properties of chaperonin systems. The results of these studies are expected to give insight into the fundamental process of cytosolic protein folding and may be useful for a better understanding of disease-related abnormal protein functions.
| Kusmierczyk, Andrew R; Martin, Jorg (2003) Nucleotide-dependent protein folding in the type II chaperonin from the mesophilic archaeon Methanococcus maripaludis. Biochem J 371:669-73 |
| Martin, Jorg (2002) Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding. Biochemistry 41:5050-5 |
| Kusmierczyk, A R; Martin, J (2001) Chaperonins--keeping a lid on folding proteins. FEBS Lett 505:343-7 |
| Kusmierczyk, A R; Martin, J (2000) High salt-induced conversion of Escherichia coli GroEL into a fully functional thermophilic chaperonin. J Biol Chem 275:33504-11 |
| Martin, J (2000) Group II chaperonins as mediators of cytosolic protein folding. Curr Protein Pept Sci 1:309-24 |
| Martin, J (1998) Role of the GroEL chaperonin intermediate domain in coupling ATP hydrolysis to polypeptide release. J Biol Chem 273:7351-7 |
