Modular structure of the trigger factor required for high activity in protein folding

Area of Science:

• Molecular Biology
• Protein Folding
• Enzymology

Background:

• Escherichia coli trigger factor (TF) is a peptidyl-prolyl cis/trans isomerase (PPIase) crucial for protein folding.
• TF associates with nascent polypeptides and the chaperone GroEL, utilizing regions beyond its catalytic domain for efficient refolding.

Purpose of the Study:

• To investigate the modular structure of E. coli trigger factor.
• To determine the contribution of individual domains (N, M, C) to the protein's folding activity.

Main Methods:

• Production and characterization of protein fragments encompassing individual domains or domain combinations (N, M, C, NM, MC).
• Analysis of fragment stability, structure, and urea-induced unfolding.
• Circular dichroism (CD) spectroscopy to assess native protein structure.

Main Results:

• TF fragments (N, M, C) are stably folded with cooperative unfolding transitions, suggesting a modular structure.
• The native TF's CD spectrum is the sum of its fragments' spectra, reinforcing the modularity hypothesis.
• High folding activity was not restored by simple N- or C-terminal fusions to the catalytic domain but was partially regained by complementary functional interaction between NM and MC constructs.

Conclusions:

• E. coli trigger factor possesses a modular structure comprising three largely independent folding units.
• Mutual stabilization exists between these units in the intact protein.
• Functional complementation between overlapping fragments partially restores the high folding activity, indicating complex domain interactions.