Characterisation of the J domain amino acid residues important for the interaction of DNAJ-like proteins with HSP70 chaperones

Hennessy, Fritha (2004) Characterisation of the J domain amino acid residues important for the interaction of DNAJ-like proteins with HSP70 chaperones. PhD thesis, Rhodes University.





The 70 kDa heat shock proteins (Hsp70s) are vital for normal protein folding, as they stabilise the unfolded state of nascent polypeptides, allowing these sufficient time to attain a correct tertiary structure. Hsp70s are aided by the DnaJ-like family of proteins, which interact with Hsp70s in order to enhance the chaperone activity of these proteins. DnaJ-like proteins contain a J domain, a seventy amino acid domain consisting of four α-helices, which is defined by the presence of an invariant tripeptide of histidine, proline and aspartic acid (HPD motif). This motif is key to the interaction between DnaJ-like proteins and Hsp70s. This thesis has focused on determining the presence of other conserved residues in the J domain and their role in mediating the interaction of DnaJ-like proteins with partner Hsp70s. DnaJ-like proteins from Agrobacterium tumefaciens RUOR were isolated and used as a model system. A. tumefaciens DnaJ (Agt DnaJ) was able to replace the lack of E. coli DnaJ in an E. coli null mutant strain, however, additional A. tumefaciens DnaJ-like proteins Agt DjC1/DjlA, Agt DjC2 and Agt DjC5 were unable to complement for the lack of E. coli DnaJ. Replacement of the Agt DnaJ J domain with J domains from these proteins resulted in non-functional chimeric proteins, despite some sequence conservation. The kinetics of the basal specific ATPase activity of Agt DnaK, and its ability to have this activity stimulated by Agt DnaJ and Agt DnaJ-H33Q were also investigated. Stimulation of the ATPase activity by Agt DnaJ ranged between 1.5 to 2 fold, but Agt DnaJ-H33Q was unable to stimulate the basal ATPase activity. Conserved amino acids in the J domain were identified in silico, and these residues were substituted in the J domain of Agt DnaJ. The ability of these derivative proteins to replace E. coli DnaJ was investigated. Alterations in the HPD motif gave rise to proteins unable to complement for lack of E. coli DnaJ, consistent with literature. Agt DnaJ-R26A was unable to replace E. coli DnaJ suggesting that Arg26 could be key to the interaction with partner Hsp70s. Agt DnaJ-D59A was unable to replace E. coli DnaJ; substitutions in Asp59 have not previously been shown to impact on the function of DnaJ. Substituting Arg63 in Agt DnaJ abrogated the levels of complementation. Substitution of several structural residues was also found to disrupt the in vivo function of Agt DnaJ suggesting that the maintenance of the structural integrity of the J domain was important for function. This study has identified a number of residues critical to the structure and function of the J domain of Agt DnaJ, and potentially of general importance as molecular determinants for DnaJ-Hsp70 interaction.

Item Type:Thesis (PhD)
Uncontrolled Keywords:heat shock proteins, protein folding, proteins analysis, proteins structure, molecular chaperones
Subjects:Y Unknown > Subjects to be assigned
Divisions:Faculty > Faculty of Science > Biochemistry, Microbiology & Biotechnology
ID Code:64
Deposited By: Rhodes Library Archive Administrator
Deposited On:14 Oct 2005
Last Modified:06 Jan 2012 16:17
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