Effect of C‐terminal truncation on the molecular chaperone function of Escherichia coli trigger factor

Abstract

Trigger Factor (TF) is an important catalyst of nascent peptide folding and possesses both peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. TF has a modular structure, containing three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding and the M domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear and the residues or regions directly involved in substrate binding have not yet been identified. To examine the chaperone function of TF and its relationship with the C-terminal domain, a number of C-terminal truncated mutants were constructed, namely: TF419, TF389, TF380, TF360 and TF344, in which the C-terminal 13, 43, 52, 72 and 88 residues were deleted respectively. Co-expressions of adenylate kinase (AK) with TF and the C-terminal truncated mutants were achieved using a plasmid pBVAT that allowed expression of TF and AK in the same plasmid under separate control. Results show that the C-terminal truncated TF mutants express about the same ability in assisting AK refolding in the co-expression system and all of the C-terminal truncated TF mutants can still bind with ribosome, indicating that the C domain of trigger factor may not be essentially important for the in vivo molecular chaperone function of TF. However, the purified C-terminal truncated TF mutants reduced dramatically the ability in assisting GAPDH refolding in vitro and almost not be able to form dimer, showing that the C-terminal of TF is necessary in in vitro molecular function and is important to form dimer.