tailieunhanh - Báo cáo Y học: The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea

Shikimate kinase was chosen as a convenient representative example of the subclass of a/b proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 M urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJÆmol)1. Approximately 95% of the enzyme activity could. | Eur. J. Biochem. 269 2124-2132 2002 FEBS 2002 doi The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea Eleonora Cerasoli1 Sharon M. Kelly1 John R. Coggins1 Deborah J. Boam1 David T. Clarke2 and Nicholas C. Price1 1 Division of Biochemistry and Molecular Biology Institute of Biomedical and Life Sciences Joseph Black Building University of Glasgow Scotland UK 2Synchrotron Radiation Department CLRC Daresbury Laboratory Warrington UK Shikimate kinase was chosen as a convenient representative example of the subclass of a b proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism CD and fluorescence respectively the enzyme was fully unfolded in 4 M urea. From an analysis of the unfolding curve in terms of the two-state model the stability of the folded state could be estimated as 17 kJ-mol-1. Approximately 95 of the enzyme activity could be recovered on dilution of the urea from 4 to M. The results of spectroscopic studies indi cated that refolding occurred in at least four kinetic phases the slowest of which k s-1 corresponded with the regain of shikimate binding and of enzyme activity. The two most rapid phases were associated with a substantial increase in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. The relevance of the results to the folding of other a b domain proteins is discussed. Keywords shikimate kinase protein folding protein unfolding circular dichroism fluorescence. Despite considerable experimental and theoretical efforts over the past 30 years the mechanism by which proteins achieve their functional three-dimensional structure .

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