Logo Logo
Switch language to English
Maier, Tobias (2006): Proteome-Wide Analyis of Chaperonin-Dependent Protein Folding in Escherichia coli. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



In Escherichia coli, the cylindrical chaperonin GroEL and its cofactor GroES promote the folding of a fraction of newly synthesized polypeptide chains by acting as an Anfinsen cage. GroEL recognizes substrate proteins with its apical domains of the tetradecameric structure. Exposed hydrophobic side chains in non-native proteins interact with GroEL and bound substrates are subsequently encapsulated under the GroES lid, where they can fold in a protected environment. Despite the detailed knowledge about structural and mechanistic features of GroEL and GroES, little is known about its genuine in vivo substrate proteins. Here, the nearly complete set of GroEL interacting proteins in vivo was identified and quantified by an approach using affinity chromatography for the isolation of GroEL/GroES/substrate complexes and subsequent analysis by mass spectrometric methods. GroEL substrate proteins were analyzed with respect to their fold types and functional classes, revealing a preference for proteins which fold into the versatile TIM barrel fold to interact with GroEL. Further in vivo and in vitro experiments with individual proteins identified as GroEL substrates verified the data obtained by the proteomic approach and allowed conclusions on the usage of the other main chaperone system in E. coli: DnaK/DnaJ/GrpE. Taken together, the results culminated in the classification of GroEL interacting proteins according to their dependence on chaperones for folding. Class I proteins are largely independent of chaperones but their folding yield can be increased by chaperone interaction. Class II proteins do not refold efficiently in the absence of chaperones in vitro, but can utilize either the DnaK or the GroEL/GroES systems for folding. Class III substrates are fully dependent on GroEL. DnaK can bind class III proteins and thus prevent their aggregation, but folding is achieved only upon transfer to GroEL.