title: Unfolding kinetics of dimeric creatine kinase measured by stopped-flow small angle X-ray scattering.
abstract: The unfolding kinetics of creatine kinase (CK) in various concentrations of urea or guanidine hydrochloride (GuHCl) was investigated by small angle X-ray scattering (SAXS) using synchrotron radiation, and compared with the results obtained by stopped-flow circular dichroism and stopped-flow fluorescence. Using the three methods, the unfolding kinetics of CK fits well to a single exponential function with similar apparent rate constants, and the amplitude of the monophasic kinetics covers the entire range of the equilibrium values. The results suggest that the unfolding time-course measured by integrated SAXS intensity corresponds to the intramolecular loss of globular structure. The refolding kinetics of 8 M urea-denatured CK was monitored in a stopped-flow apparatus by following the spectroscopic changes, and the final state of folding was investigated by SAXS. A substantial part of the ellipticity is recovered within a burst phase, indicating that the secondary structure forms at an early stage in refolding. The R(g) value of the final folded state was 33.6 A when the folding buffer contained 20% glycerol, which is characteristic of native-like compactness and globularity.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Qin
authors.forename: Jun-Mei
authors.initials: JM
authors.lastname: Zhou
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kihara
mesh_terms: D002942:Circular Dichroism / Q000379:methods; D003402:Creatine Kinase / Q000737:chemistry*; D019281:Dimerization; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods; D017356:Synchrotrons; D013997:Time Factors; D014508:Urea / Q000737:chemistry; D014965:X-Rays
chemical_list: D014508:Urea; D003402:Creatine Kinase