Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form

Area of Science:

• * Molecular Biology
• * Cell Biology
• * Protein Biochemistry

Background:

• * Pro-OmpA, a precursor protein, can assemble into bacterial inner membrane vesicles with ATP and NADH.
• * Purification of pro-OmpA is necessary to identify proteins involved in its membrane assembly.
• * In vitro synthesized pro-OmpA requires additional factors for efficient membrane insertion.

Purpose of the Study:

• * To identify soluble proteins required for pro-OmpA membrane assembly.
• * To investigate the role of a potential protein factor in pro-OmpA folding and assembly.
• * To characterize the properties of the identified soluble factor.

Main Methods:

• * Immunoaffinity purification of [35S]pro-OmpA using anti-OmpA antibodies.
• * Anion-exchange chromatography in 8 M urea for pro-OmpA purification.
• * Assembly assays with purified pro-OmpA, Escherichia coli cytoplasmic fraction (S100), and dialysis.
• * Sedimentation velocity studies to determine trigger factor molecular weight.

Main Results:

• * Purified pro-OmpA, after dialysis, was soluble but incompetent for membrane assembly.
• * Addition of S100 to pro-OmpA prior to dialysis restored assembly competence, indicating a soluble factor ('trigger factor').
• * Trigger factor did not bind to the anti-OmpA column when pro-OmpA was acid-denatured, suggesting it associates with pro-OmpA.
• * Sedimentation velocity indicated trigger factor has an apparent molecular weight of ~60,000.

Conclusions:

• * A soluble factor, termed 'trigger factor,' is crucial for the translocation-competent folding of pro-OmpA.
• * Trigger factor facilitates the proper folding of pro-OmpA as urea is removed during purification.
• * This factor likely plays a role in the assembly of other precursor proteins into bacterial membranes.