Effect of C-terminal truncation on the molecular chaperone function and dimerization of Escherichia coli trigger factor.
To examine the role of the C-terminal domain in the chaperone function of trigger factor (TF), a number of truncation mutants were constructed, namely: TF419, TF389, TF380, TF360, TF344, and TF251, in which the C-terminal 13, 43, 52, 72, 88 residues or the entire C-domain were deleted, respectively. Co-expression of mutant chicken adenylate kinase (AK) with TF and the C-terminal truncation mutants was achieved using a plasmid pBVAT that allows expression of TF and AK from a single plasmid. The results show that truncation of the C-terminus of TF has only minor effect on its ability to assist AK refolding in vivo. Further, ribosome-binding experiments indicate that C-terminal truncation mutants can still bind to the ribosome and the presence of the C-terminus may in fact lower the affinity of TF for the ribosome in vivo. This indicates that the C-domain of trigger factor may not be essential for the ribosome-associated molecular chaperone function of TF. However, the purified TF C-terminal truncation mutants had a dramatically reduced ability to assist rabbit muscle GAPDH refolding in vitro and a reduced tendency to dimerize. This shows that the structural integrity of the C-terminus contributes to both the chaperone function of TF and the stability of the dimeric form.
Zeng
Li-Ling
LL
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Yu
Ling
L
Li
Zhen-Yu
ZY
Perrett
Sarah
S
Zhou
Jun-Mei
JM
D000263:Adenylate Kinase / Q000378:metabolism; D000818:Animals; D002645:Chickens; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011817:Rabbits; D017384:Sequence Deletion / Q000235:genetics
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D000263:Adenylate Kinase; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase