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Hirtreiter, Angela Maria (2007): Protein Folding in Archaea: Analysis of the co-existing Group I and Group II chaperonins in M. mazei. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Chaperonins are a specific class of barrel-shaped chaperones, present in almost all organisms. Newly synthesized proteins encapsulated by the chaperonin can attain their native structure unimpaired by aggregation during repeated cycles of ATP-dependent binding and release. Chaperonins are generally divided into two groups. Group I chaperonins, such as the barrel-shaped GroEL oligomer, are found predominantly in bacteria and cooperate with cofactors of the Hsp10 familly (i.e. GroES). The Group II chaperonins, on the other hand, do not require a Hsp10- cofactor and are found in the eukaryotic cytosol and in archaea. The function of GroEL is understood in great detail and the substrate interaction proteome has been recently identified. In contrast, our knowledge about the natural substrates of Group II chaperonins is deficient and as a consequence, mechanistical studies on Group II chaperonins have been limited to using the eukaryotic model substrates actin and tubulin as well as heterologous model substrates. In the present study, the complete substrate spectrum of a Group II chaperonin, the thermosome (Ths) of the mesophilic archaeon Methanosarcina mazei (M. mazei), was analysed for the first time. In addition, the unique coexistence of both the goup I and the group II chaperonins in M. mazei, which was confirmed in the initial part of the study, provided the opportunity to obtain new insights into how the substrate selection differs between the two chaperonin groups. For these purposes, the chaperonin substrates were isolated by immunoprecipitation of the chaperonin-substrate complexes and identified by liquid chromatography coupled mass spectrometry (LC-MS) using three different approaches: LC-MS after separation of the proteins (i) by classical 2D-PAGE, (ii) by difference gel electrophoresis (Ettan DIGE) and (iii) by 1D-PAGE. Analysis of substrates of both the thermosome (MmThs) and GroEL/GroES (MmGroEL, MmGroES) of M. mazei revealed that each chaperonin handles a defined set of substrates, and both chaperonins contribute to the folding of ~17% of the proteins in the archaeal cytosol. Bioinformatic analysis revealed that the chaperonin specificity is governed by a combination of a various physical properties (hydrophobicity, net charge and size), structural features (i.e. the domain fold), and less concrete characteristics like the evolutionary status and, in this context, the phylogenetic origin of the substrate.