The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm.
PreOmpA-bovine pancreatic trypsin inhibitor (BPTI) (Goldenberg, D. P. (1988) Biochemistry 27, 2481-2489) was expressed in Escherichia coli, and the folding pathway of the mature protein in the periplasmic space was analyzed by pulse-chase experiments. Folding intermediates were trapped with iodoacetamide, immunoprecipitated with antisera specific for either the reduced or the native protein, and resolved by electrophoresis. In vivo, native BPTI formed with a half-life of 7 min which is 3-fold faster than the optimal in vitro folding rate in growth media supplemented with low molecular weight disulfides. The measured in vivo half-life includes the time required for translocation and processing by leader peptidase and therefore represents the lower limit for the actual folding rate in the cell. In addition to the native species, two-disulfide intermediates accumulated in the cell at appreciable levels and did not chase to the native species for at least 20 min. We found that the folding of BPTI in E. coli was absolutely dependent on DsbA, a protein which accelerates the formation of disulfide bonds in the periplasm. In a dsbA mutant strain, trace amounts of oxidized BPTI could be detected only in cultures grown under strongly oxidizing conditions. In wild-type cells, the addition of different concentrations of GSH/GSSG or oxidized dithiothreitol did not affect the kinetics of BPTI oxidation by DsbA. Finally, even though the folding of BPTI in vitro decreases by almost 10-fold/unit pH decrease, folding in cells grown at pH 6.0 was only marginally slower than folding in cells grown at neutral pH, despite the fact that the pH of the periplasmic space varies in response to the extracellular fluid.
Ostermeier
M
M
Department of Chemical Engineering, University of Texas, Austin 78712.
Georgiou
G
G
D000818:Animals; D007611:Aprotinin / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003593:Cytoplasm; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D017510:Protein Folding
D007611:Aprotinin