PMID:10446363
| Citation |
Michaels, JE, Shiba, K and Miller, WT (1999) Autonomous folding of a C-terminal inhibitory fragment of Escherichia coli isoleucine-tRNA synthetase. Biochim. Biophys. Acta 1433:103-9 |
|---|---|
| Abstract |
We previously reported that C-terminal fragments of Escherichia coli Ile-tRNA synthetase, a monomeric enzyme of 939 amino acids, act as dominant negative inhibitors of the wild-type enzyme in vivo and in vitro. Our experiments suggested that it is possible to block the functional assembly of a monomeric protein by interfering with the folding pathway. We postulated that the inhibitory C-terminal fragments fold autonomously, and in the presence of full-length Ile-tRNA synthetase, trap the N-terminal portion of polypeptide in an unproductive complex. Here, we report the results of experiments aimed at understanding the mechanism of dominant negative inhibition. We have carried out biophysical experiments on fragment 585-939 of Ile-tRNA synthetase, which we previously determined to be the minimal inhibitory unit. Circular dichroism and fluorescence spectroscopy indicate that this fragment forms a compact and stable structure in solution. The secondary structure of this fragment is predominantly alpha-helical, consistent with the crystal structure of Ile-tRNA synthetase from another organism. The C-terminal fragment is capable of forming native-like secondary and tertiary structure after refolding from guanidine HCl. Taken together, the results are consistent with the hypothesis that the inhibitory fragment of Ile-tRNA synthetase forms an independent folding unit. |
| Links | |
| Keywords |
Circular Dichroism; Enzyme Stability; Escherichia coli/enzymology; Hot Temperature; Isoleucine-tRNA Ligase/antagonists & inhibitors; Isoleucine-tRNA Ligase/chemistry; Peptide Fragments/chemistry; Protein Denaturation; Protein Folding; Spectrometry, Fluorescence |
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