Communication between the domains of the DnaK chaperone characterized by single molecule force spectroscopy
Zusammenfassung der Projektergebnisse
In order to observe conformational changes in DnaK at the single molecule level, I have prepared several variants of full-length DnaK and its individual domains for single molecule force spectroscopy. Stretching curves of the single molecule DnaK displayed two unfolding events corresponding to the unfolding of the SBD and NBD. The SBD unfolding proceeds through two transitions corresponding to the C-terminal helices and the lid helix together with β-subdomain whereas NBD unfolds cooperatively in a single step. The mechanical stabilities of domains and folding/unfolding kinetics in the absence of ligands resemble very closely the properties of the isolated domains. In the presence of Mg2+ATP, however, the disruption of the otherwise mechanically stable interface between the lid and β-subdomain of the SBD results in the higher folding/unfolding dynamics. Such rapid fluctuations were not observed in the presence of Mg2+ADP. Thus, real-time observation of force fluctuations of the lid conformation provides a sensitive single-molecule monitor of the ATP state of DnaK. The NBD unfolds highly cooperatively through discrete transiently populated unfolding intermediates whereas refolding is gradual with at least four intermediates; two en route refolding intermediates are well populated under load. The structure of these refolding intermediates constitutes nearly complete nucleotide binding pocket and they bind Mg2+ATP and ADP. The other intermediates populate only at very low forces <2 pN. Comparison of the bacterial DnaK with mitochondrial NBD shows that unfolding pathways of the bacterial and mitochondrial NBD are nearly identical; but refolding pathways of the both proteins have diverged dramatically. Surprisingly, in a few cases the C-terminal flexible linker of DnaK (604-638) can bind to the peptide binding site and unfolding is highly cooperative. In the most cases, SBD unfolds gradually through two transitions. The helical bundle sub-domain with a part of the helical lid folds/unfolds relatively fast. The folding properties of the helical parts are modulated by the presence of the folded β-subdomain and the rest of the lid helix. The folded β-subdomain refolds very slowly and shows clear dependence on the previous folding status. A possible role in this memory effect can be ascribed to the cis proline bonds presented in the folded state of the SBD.
Projektbezogene Publikationen (Auswahl)
- Force as a single molecule probe of multidimensional protein energy landscapes. Curr Opin Struct Biol. 2012 Dec 29. [Epub ahead of print]
Zoldák G, Rief M
(Siehe online unter https://doi.org/10.1016/j.sbi.2012.11.007)
