An 8-fold {beta}{alpha} barrel protein with redundant folding possibilities |
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Authors: | Luger Karolin; Szadkowski Halina; Kirschner Kasper |
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Affiliation: | Abteilung Biophysikalische Chemie, Biozentrum der Universität Basel Klingelbergstrasse 70, CH-4056 Basel, Switzerland |
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Abstract: | Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß )10proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß )8 protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß )10variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß )8 protein. The datasuggest that the folding of the (ß )10 variant is controlledthermodynamically both in vivo and in vitro. |
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Keywords: | ß barrel/" target="_blank">gif" ALT="{alpha}" BORDER="0"> barrel/ circular permutation/ protein folding/ terminal repeat/ TIM barrel |
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