title: The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea.
abstract: Shikimate kinase was chosen as a convenient representative example of the subclass of alpha/beta 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 0.36 m. The results of spectroscopic studies indicated that refolding occurred in at least four kinetic phases, the slowest of which (k = 0.009 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 alpha/beta domain proteins is discussed.
authors: Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Scotland, UK.
authors: Eleonora
authors: E
authors: Cerasoli
authors: Sharon M
authors: SM
authors: Kelly
authors: John R
authors: JR
authors: Coggins
authors: Deborah J
authors: DJ
authors: Boam
authors: David T
authors: DT
authors: Clarke
authors: Nicholas C
authors: NC
authors: Price
mesh_terms: D002942:Circular Dichroism; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D005453:Fluorescence; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D014508:Urea / Q000494:pharmacology
chemical_list: D014508:Urea; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase