Unraveling multistate unfolding of rabbit muscle creatine kinase

GdmCl-induced unfolding of rabbit muscle creatine kinase, CK, has been studied by a variety of physico-chemical methods including near and far UV CD, SEC, intrinsic fluorescence (intensity, anisotropy and lifetime) as well as intensity and lifetime of bound ANS fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of ‘phase diagram’, i.e. I_λ1 versus I_λ2 dependence, where I_λ1 and I_λ2 are fluorescence intensity values measured on wavelengths λ₁ and λ₂ under the different experimental conditions for a protein undergoing structural transformations. The unfolding behavior of CK was shown to be strongly affected by association of partially folded intermediates. A model of CK unfolding, which takes into account both structural perturbations and association of partially folded intermediates has been elaborated.