TY  - GEN
KW  - in vitro 
KW  -  co-translational 
KW  -  TRiC 
KW  -  Aktinprotein 
KW  -  folding 
KW  -  chaperone 
KW  -  actin 
KW  -  co-translational
Y1  - 2007///
AV  - public
ID  - heidok7793
TI  - Analysis of chaperone function in multi-domain protein folding
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/7793/
N2  - Proteins are the central molecules of life. To become functionally active, newly synthesized polypeptide chains must fold into unique three-dimensional structures on a biologically relevant time scale. Although the information required for correct folding resides in the linear amino acid sequence of a protein, execution of the folding process under cellular conditions is critically dependent on the assistance of ?helper proteins? termed molecular chaperones. This group of proteins shares the common function of binding to newly synthesized non-native proteins to prevent off-pathway reactions which otherwise would lead to misfolding and aggregation. Two major classes of chaperones, the Hsp70s and the chaperonins, have been implicated in de novo protein folding in the cytosol. While Hsp70s are primarily involved in stabilizing nascent chains until a complete domain has emerged from the ribosome and is competent for folding, the barrel-shaped chaperonins provide physically defined compartments inside which complete proteins or protein domains can fold unimpaired by aggregation. Proteins of eukaryotic origin which, on average, have a more complex architecture than their prokaryotic counterparts, often fold inefficiently upon expression in bacterial hosts. For many decades, this phenomenon placed great limitations on the recombinant production of proteins.  In the present study, eukaryotic multi-domain proteins were synthesized in cell-free translation systems in order to investigate the contribution of individual chaperones to their de novo folding: Upon expression under chaperone depleted conditions in an Escherichia coli based lysate, the modular eukaryotic protein firefly luciferase was demonstrated to fold by a rapid default pathway, tightly coupled to translation. However, only a minor fraction of the translated protein chains folded correctly. In contrast, supplementation of the lysate with purified trigger factor and DnaK/DnaJ/GrpE increased the amount of native protein, but markedly delayed firefly luciferase folding relative to translation. Interestingly, while the bacterial multi-domain protein ß-galactosidase uses the endogenous chaperone machinery effectively (Agashe et al., 2004), the efficient co-translational domain folding of firefly luciferase observed in eukaryotes is not compatible with the prokaryotic chaperone system. Thus, important differences between bacterial and eukaryotic cells seem to exist in the coupling of translation and folding. Moreover, the bacterial lysate which did not contain any eukaryotic chaperones was further utilized to determine the minimum requirements for the folding of newly synthesized actin. By conducting in vitro translation reactions in the presence of purified components, the chaperonin TRiC was found to be the only eukaryotic chaperone absolutely necessary and sufficient for de novo actin folding. The actin thus produced bound DNase I and polymerized into filaments, hallmarks of its native state. Lysate supplementation with the bacterial chaperonin GroEL/GroES or the DnaK/DnaJ/GrpE chaperones led to mostly soluble actin, yet failed to facilitate its correct folding. Notably, actin folding in the TRiC-supplemented bacterial lysate occurred with slower kinetics when compared to the eukaryotic cytosol. Additionally, TRiC was demonstrated to be capable of mediating the domain-wise folding of modular proteins by their partial encapsulation in the chaperonin cavity. This was based on the fact that upon expression of actin multi-domain fusion proteins both in vivo and in vitro, the proteins properly integrated into yeast cytoskeleton structures as well as formed stable complexes with DNase I. The mechanism of how TRiC might promote the folding of individual protein domains is thereby reminiscent of the domain-wise endoproteolytical degradation of proteins by the proteasome, as reported recently (Liu et al., 2003). 
A1  - Stemp, Markus Johann
ER  -