title: An equilibrium study of the dependence of secondary and tertiary structure of creatine kinase on subunit association.
abstract: Several physical properties of dimeric creatine kinase in increasing concentrations of guanidine hydrochloride (GDN/HCl) were evaluated and correlated with degree of subunit dissociation, determined by isozyme competitive hybridization. Three distinct stages were observed that correlated with phases before, during and after dissociation. In 0.2 M GDN/HCl, before significant dimer dissociation, creatinine kinase has 75% of its original activity, and exhibits only small decreases in circular dichroism, intrinsic fluorescence and emission maximum. The spectral characteristics of creatine kinase (CK) derivatized with 5-[(((2-iodoacetyl)amino)ethyl)amino]naphthalene-1- sulfonate (AEDANS-CK) at the cysteine near the active site are suggestive of a slightly more non-polar environment. A decrease in steady-state anisotropy (0.140 to 0.132) was characterized by time-resolved methods. The slow component of the time-resolved anisotropy decay law, which reflects global protein rotation, is decreased only from 36.6 to 33.4 ns. The faster component decreases from 1.95 to 0.74 ns which suggests the active-site domain is more sensitive to conformation perturbation than the protein as a whole. Overall these observations suggest the subunits within the dimeric state are rather stable in dilute denaturant, but undergo a minor contraction in conformation. The region of the active site, as reported by the extrinsic fluorophore, is less polar but apparently more flexible in dilute denaturant. Between 0.5 M and 1 M GDN/HCl, most of the dimers dissociate, 63% of helical content is lost and inactivation is complete. The intrinsic fluorescence shifts 8 nm to the red and increases by 35%, indicating exposure of tryptophans to solvent and release of quenching, perhaps between residues on separate subunits. Over the same denaturant range, the spectral characteristics and lifetime of AEDANS-CK suggests less exposure of the active site to solvent. Time-resolved anisotropy measurements show that the sharp decrease in steady-state anisotropy to 0.086 is due to a decrease in macromolecular rotation to 22 ns. This may represent the rotational correlation time of a relatively intact subunit, and suggests limited subunit unfolding accompanying dissociation. Dissociation is complete in 1.5 M GDN/HCl. The subunits still retain 20% helical content in 2 M denaturant and not until 5 M GDN/HCl is all helical structure eliminated. Above 2 M GDN/HCl, AEDANS-CK exhibits sharp decreases in steady-state anisotropy, fluorescence lifetime and the long-lived component in the time-resolved anisotropy decay law. These results reveal a catastrophic loss of tertiary structure by the subunits and may define the physical properties of the random coil.
authors.affiliation: Department of Chemistry, University of South Florida, Tampa 33620.
authors.forename: S H
authors.initials: SH
authors.lastname: Grossman
mesh_terms: D000818:Animals; D016880:Anisotropy; D002384:Catalysis; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D009282:Naphthalenesulfonates; D012997:Solvents; C006421:1,5-I-AEDANS; D003402:Creatine Kinase