title: Folding of staphylococcal nuclease A studied by equilibrium and kinetic circular dichroism spectra.
abstract: The urea-induced unfolding of staphylococcal nuclease A has been studied by circular dichroism both at equilibrium and by the kinetics of unfolding and refolding (pH 7.0 and 4.5 degrees C), as a function of Ca2+ and thymidine 3',5'-diphosphate (pdTp) concentration. The results are as follows. (1) The unfolding transition is shifted to higher concentrations of urea by Ca2+ and pdTp, and the presence of both ligands further stabilizes the protein. (2) In the first stage of kinetic refolding, the peptide ellipticity changes rapidly within the dead time of stopped-flow measurement (15 ms), indicating accumulation of a transient intermediate. This intermediate is remarkably less stable than those of other globular proteins previously studied. (3) Dependence of the folding and unfolding rate constants on urea concentration indicates that the critical activated state of folding ("transition state") has considerable structural organization. The transition state does not, however, have the capacity to bind Ca2+ and pdTp, as indicated by the effects of these ligands on the unfolding rate constant. (4) There are at least four different phases in the refolding kinetics in native conditions below 1 M urea. In the absence of pdTp, there are two phases in unfolding, while in the presence of pdTp the unfolding kinetics show a single phase. Some characteristics of the transient intermediate and of the transition state for folding are discussed.
authors.affiliation: Department of Polymer Science, Faculty of Science, Hokkaido University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Sugawara
authors.forename: K
authors.initials: K
authors.lastname: Kuwajima
authors.forename: S
authors.initials: S
authors.lastname: Sugai
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013210:Staphylococcus / Q000201:enzymology*
chemical_list: D008836:Micrococcal Nuclease