Heat-shock proteins

Under a situation of stress, different classes of chaperones/Hsps (heat-shock protein) perform complementary roles in proteic folding. Some of them, for example small Hsp, sHsp and Hsp70, acts keeping the proper protein folding and avoiding aggregation. In this way, they keep the protein in a proper state for the action of refolding, which is done by other chaperones. When denatured or incorrect folded proteins form aggregates, they are solubilized by Hsp100/Clp, followed by refolding or degradation by proteases (ARSENE et al., 2000). Hsps are highly conserved between species, therefore, in this project; we seek the overexpression of Hsps from Escherichia coli, which will be described next.

ClpB

The bound of ClpB to a protein stimulates is ATPase activity. The ATP and external proteins hydrolysis undo denatured protein clumps. With this, new hydrophobic sites are exposed which contribute to the bound and action of chaperones responsible for solubilization and refolding. ClpB exists in two isoforms, long ClpB (alpha) [ClpB93] and short ClpB (beta) [ClpB79], which is started by an internal initiation codon GUG at position 149 (GOTTESMAN et al., 1990). The coordinated expression of both forms showed necessary to protect cells from temperature shift death (BARNETT et al., 2000).

DnaK

DnaK is composed by two domains, the N-terminal nucleotide binding domain (NBD) and the C-terminal substrate binding domain. The bound and hydrolysis of ATP cause great conformational changes on DnaK. The closed form containing ADP has stable interactions with substrate, opposite to the opened form containing ATP. It was demonstrated as well in vitro as in vivo the cooperation between ClpB and DnaK to disaggregate and reactivate insoluble protein clumps (GLOVER et al., 1998).

IbpA/IbpB

sHsps (small heat shock proteins) are molecular chaperones that binds to still not folded proteins, keeping them in a proper state to be folded. Two elements of this family, IbpA and IbpB are present in E. coli. The cooperation between these two molecules is essential to avoid proteic aggregation (STÓZECKA et al., 2012). By their disposal as an operon, and acting together, they will be cloned together. The original sequence from E. coli does not have restriction sites for the restriction enzymes necessary to the assembly pattern.

Process and application

The second module of this project corresponds to the overexpression of chaperones during cellular growth, which characterizes an important tool to solubilize proteins, in a way that together with the process described in the first module it will present a favorable situation to correctly express the limonene synthase by the promoter PuspA.

References

ARSENE, F.; TOMOYASU, T.; BUKAU, B. The heat shock response of Escherichia coli. Int J Food Microbiol. Apr 2000.

GOTTESMAN S., SQUIRES C. PICHERSKY E., CARRINGTON M., HOBBS M., MATTICK J., DALRYMPLE B., KURAMITSU H., SHIROZA T., FOSTER T., CLARK W., ROSS B. AND MAURIZI M. Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes. Proc. Natl. Acad. Sci. USA 87, 1990;

BARNETT M. E., ZOLKIEWSKA A., ZOLKIEWSKI M. Structure and Activity of ClpB from Escherichia coli: Role of the Amino- and Carboxy-terminal Domains. J Biol Chem. Author manuscript; available in PMC 2007 Mar 9. Published in final edited form as: J Biol Chem; 275(48): 37565–37571. Dec 2000;

GLOVER J. R., LINDQUIST S. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell; 94:73–82; 1998;

STRÓZECKA J., CHRUSCIEL E., GÓRNA E., SZYMANSKA A., ZIĘTKIEWICZ S., LIBEREK K. Importance of N- and C-terminal regions of IbpA, Escherichia coli small heat shock protein, for chaperone function and oligomerization. J Biol Chem. 20 jan 2012.