CONICET | Buscador de Institutos y Recursos Humanos

The mechanism by which Hsp70 chaperones assist the folding of non-native substrates is still unclear. In particular, it is yet unknown how non-native proteins are re-folded. Two alternative mechanisms can be imagined. In the first one, Hsp70 plays a rather passive role. Through repetitive substrate binding and release cycles, the free concentration of the substrate is kept low enough to prevent aggregation while allowing free molecules to fold to the native state. In the second one, the binding and release cycles induce local conformational changes in the substrate by using the energy of ATP, which permits to overcome kinetic barriers for folding to the native state. In order to get a better understanding of the molecular mechanism of Hsp70, the conformation of substrates and possible conformational changes due to the action of Hsp70 have to be probed. Hsp70 chaperones assist a large variety of protein folding processes in the cell, such as de novo protein synthesis, protein translocation, assembly and disassembly of protein complexes, and protein misfolding, especially under heat stress conditions. Hsp70 proteins control, in addition, cellular processes by transient connection to specific, folded proteins. Thus, Hsp70 proteins bind to numerous proteins regulating the biological activity of the cell. One of those substrates is the replication initiator protein RepE. The DnaK-dependent activation of RepE is essential for replication initiation of mini-F plasmids. This activation is accompanied by the conversion of stable RepE dimers into monomers.The purpose of this work is to study DnaK-substrate interactions with RepE to gain new insights about the molecular mechanism of the E. coli DnaK chaperone. To achieve this, we used amide hydrogen exchange techniques. By comparing the amide hydrogen exchange behavior of dimeric RepE wild-type with the monomeric mutant RepE54, the dimerization interface was identified. In addition, significant conformational changes were detected that occur upon monomerization and localized to two regions in the three-dimensional structure. The interaction between DnaK and RepE has been studied using H/D footprinting and BIACORE.

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