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PMID:10748201
| Citation |
Ramm, K and Plückthun, A (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro. J. Biol. Chem. 275:17106-13 |
|---|---|
| Abstract |
We recently identified FkpA by selecting for the increased yield of antibody single-chain Fv (scFv) fragments in phage display, even of those not containing cis-prolines. We have now investigated the properties of FkpA in vitro. The peptidylprolyl cis-trans-isomerase activity of FkpA was found to be among the highest of any such enzyme with a protein substrate, yet FkpA is not able to enhance the proline-limited refolding rate of the disulfide-free hu4D5-8 scFv fragment, probably due to inaccessibility of Pro-L95. Nevertheless, the yield of the soluble and functional scFv fragment was dramatically increased in vitro in the presence of FkpA. Similar effects were observed for an scFv fragment devoid of cis-prolines. We are thus forced to conclude that the observed folding-assisting function is independent of the isomerase activity of the protein. The beneficial effect of FkpA was found to be due to two components. First, FkpA interacts with early folding intermediates, thus preventing their aggregation. Additionally, it has the ability to reactivate inactive protein, possibly also by binding to a partially unfolded species that may exist in equilibrium with the aggregated form, which may thus be released on a productive pathway. These in vitro measurements therefore fully reflect the in vivo results from periplasmic overexpression of FkpA. |
| Links |
PubMed Online version:10.1074/jbc.M910234199 |
| Keywords |
Enzyme Stability; Escherichia coli/enzymology; Escherichia coli Proteins; Immunoglobulin Fragments/immunology; Immunoglobulin Fragments/metabolism; Immunophilins/chemistry; Immunophilins/metabolism; Kinetics; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Molecular Chaperones/metabolism; Peptidylprolyl Isomerase; Periplasm/enzymology; Proline/metabolism; Protein Folding; Thermodynamics |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0003755: peptidyl-prolyl cis-trans isomerase activity |
ECO:0000314: |
F |
Figure 2: Fluorescence traces of refolding of RNase T1 (0.2 μM) at 10 °C starting from equilibrium-denatured protein (trace a) in the absence or presence of 0.5 nM (trace b), 2 nM (trace c), and 5 nM (trace d) FkpA (calculated for the dimer). The intensity at 323 nm was followed. Inset, the refolding curves were evaluated with a double exponential fit; and the rates of the faster refolding phase, divided by the uncatalyzed rate, are plotted against the respective FkpA concentration present during the measurement. arb., arbitrary. |
complete | ||||
Notes
See also
References
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