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

Journal Article · · Proc. Natl. Acad. Sci. U.S.A.; (United States)
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Pro-OmpA that is synthesized in vitro can assemble into bacterial inner membrane vesicles in the presence of ATP and NADH. The authors have purified pro-OmpA to determine which additional soluble proteins are necessary for its membrane assembly. (/sup 35/S)Pro-OmpA was bound to Sepharose-linked antibody to OmpA, then eluted with 8 M urea and chromatographed on an anion-exchange resin in 8 M urea. This pro-OmpA is purified 2000-fold and is radiochemically pure. After dialysis, it is soluble but incompetent for membrane assembly. Addition of an Escherichia coli cytoplasmic fraction (S100) to the assembly reaction does not allow translocation. However, when S100 is added to pro-OmpA prior to dialysis, full assembly competence is restored, suggesting that a soluble factor, termed trigger factor, triggers the folding of pro-OmpA into an assembly-competent form as the urea is removed. They noted that, prior to the last purification step, the immunoaffinity-purified pro-OmpA was partially competent for membrane assembly without addition of trigger factor. To test whether trigger factor had bound to the antibody column by means of its association with pro-OmpA, the crude pro-OmpA was acid-denatured prior to immunoadsorption. In this experiment, the trigger factor did not bind to the anti-OmpA column, and S100 was required for renaturation of this (/sup 35/S)pro-OmpA. As suggested by this experiment, the crude (/sup 35/S)pro-OmpA was in complex with other proteins. Sedimentation velocity studies showed that the trigger factor has an apparent molecular weight of approx. = 60,000. They propose that it is required for translocation-competent folding of pro-OmpA and other precursor proteins.
Research Organization:
Univ. of California, Los Angeles (USA)
OSTI ID:
5209714
Journal Information:
Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 84:15; ISSN PNASA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ATP
AUTORADIOGRAPHY
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOSYNTHESIS
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHROMATOGRAPHY
CYSTEINE
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
IN VITRO
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ION EXCHANGE CHROMATOGRAPHY
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MEMBRANE TRANSPORT
MEMBRANES
MICROORGANISMS
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PROTEINS
PURIFICATION
RADIOISOTOPES
SEDIMENTATION
SEPARATION PROCESSES
SULFUR 35
SULFUR ISOTOPES
SYNTHESIS
THIOLS