MODULAR STRUCTURE OF THE TRIGGER FACTOR REQUIRED FOR HIGH-ACTIVITY INPROTEIN-FOLDING

The Escherichia coli trigger factor is a peptidyl-prolyl cis/trans iso merase (PPIase) which catalyzes proline-limited protein folding extrem ely well. It has been found associated with nascent protein chains as well as with the chaperone GroEL. The trigger factor utilizes protein regions outside the central catalytic domain for catalyzing refolding of unfolded proteins efficiently. Here we produced several fragments w hich encompass individual domains or combinations of the middle FKBP-l ike domain (M) with the N-terminal (N) and C-terminal (C) regions, res pectively. These fragments appear to be stably folded. They show order ed structure and cooperative urea-induced unfolding transitions, and t he far-UV CD spectrum of the intact trigger factor is well represented by the sum of the spectra of the fragments. This suggests that the na tive trigger factor shows a modular structure, which is composed of th ree fairly independent folding units. In the intact protein there is a slight mutual stabilization of these units. The high enzymatic activi ty in protein folding could not be restored by fusing alternatively th e N or the C-terminal regions to the catalytic. domain (in NM and MC c onstructs, respectively). Surprisingly, the high folding activity of t he intact trigger factor has been regained partially by functional com plementation of the overlapping NM and MC constructs. (C) 1997 Academi c Press Limited.