Unfolding of rabbit muscle creatine kinase induced by acid. A study using electrospray ionization mass spectrometry, isothermal titration calorimetry, and fluorescence spectroscopy.
Electrospray ionization mass spectrometry, isothermal titration calorimetry (ITC), fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE have been used to study the unfolding of rabbit muscle creatine kinase (MM-CK) induced by acid. The mass spectrometric experiments show that MM-CK is unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. The dimeric protein becomes an equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) between pH 5.0 and 3.0 and is almost fully unfolded at pH 3.0 reached. The results from a "phase diagram" method of fluorescence show that the conformational transition between the native state and the intermediate of MM-CK occurs in the pH range of 7.0-5.2, and the transition between the intermediate and the unfolded state of the protein occurs between pH 5.2 and 3.0. The intrinsic molar enthalpy changes for formation of the unfolded state of MM-CK induced by acid at 15.0, 25.0, 30.0, and 37.0 degrees C have been determined by ITC. A large positive molar heat capacity change of the unfolding, 8.78 kcal mol-1 K-1, at all temperatures examined indicates that hydrophobic interaction is the dominant driving force stabilizing the native structure of MM-CK. Combining the results from these four methods, we conclude that the acid-induced unfolding of MM-CK follows a "three-state" model and that the intermediate state of the protein is a partially folded monomer.
Liang
Yi
Y
College of Life Sciences, Wuhan University, Wuhan 430072, China. liangyi@whu.edu.cn
Du
Fen
F
Sanglier
Sarah
S
Zhou
Bing-Rui
BR
Xia
Yi
Y
Van Dorsselaer
Alain
A
Maechling
Clarisse
C
Kilhoffer
Marie-Claude
MC
Haiech
Jacques
J
D000085:Acetates / Q000494:pharmacology; D000143:Acids / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002151:Calorimetry; D003402:Creatine Kinase / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000494:pharmacology; D005976:Glutaral / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D000085:Acetates; D000143:Acids; D000817:Anilino Naphthalenesulfonates; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D003402:Creatine Kinase; D009113:Muramidase; C018824:ammonium acetate; D005976:Glutaral