Unraveling Multistate Unfolding of Rabbit Muscle Creatine Kinase

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Molten Globule, Pre-molten Globule, Creatine Kinaseintrinsic Fluorescence, Partially Folded Intermediate

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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. Iλ1 versus Iλ2 dependence, where Iλ1 and Iλ2 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.

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Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, v. 1596, issue 1, p. 138-155