Multiple-state equilibrium unfolding of guanidino kinases.
The denaturant-induced equilibrium unfolding of octameric mitochondrial creatine kinase, dimeric cytosolic muscle-type creatine kinase, and monomeric arginine kinase was investigated. Stable unfolding intermediates for all three enzymes were manifested by a strongly biphasic red shift of intrinsic protein fluorescence upon increasing denaturant concentrations. In the intermediate state, all proteins were monomeric and enzymatically inactive, but still retained a globular shape. Native tertiary structure interactions were largely disrupted, while at least 50% of the secondary structures were conserved, as suggested by near- and far-UV circular dichroism, respectively. A significantly increased surface hydrophobicity of the intermediate conformation, compared to both the native and the fully unfolded states, was observed by the binding of the hydrophobic fluorescent dye ANS. The observed properties agree formally with the definition of the molten globule state, but can be alternatively explained by a sequential unfolding of individual domains, involving a transient exposure of domain interfaces. Very similar unfolding profiles for all three proteins suggest that the formation of stable unfolding intermediates is not a consequence of the specific oligomeric structures of the CKs but rather due to a common, probably two-domain architecture of the guanidino kinase protomers.
Gross
M
M
Institute for Cell Biology, Swiss Federal Institute of Technology, Zürich.
Lustig
A
A
Wallimann
T
T
Furter
R
R
D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry*; D003600:Cytosol / Q000201:enzymology; D019791:Guanidine; D006146:Guanidines; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea
D006146:Guanidines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014508:Urea; D003402:Creatine Kinase; D001122:Arginine Kinase; D019791:Guanidine