Koshiba, Takumi and Kobashigawa, Yoshihiro and Demura, Makoto and Nitta, Katsutoshi (2001) Energetics of three-state unfolding of a protein: canine milk lysozyme. Protein Engineering, 14 (12). pp. 967-974. ISSN 0269-2139. http://resolver.caltech.edu/CaltechAUTHORS:20111118-081615720
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Thermodynamics of thermal transitions of a calcium-binding lysozyme, canine milk lysozyme (CML), was studied using differential scanning calorimetry and compared with those for homologous proteins, human α-lactalbumin (α-hLA) and equine milk lysozyme (EML). The results showed that CML and EML exhibit two clear heat absorption peaks in the absence of calcium ions (apo-form), although the cooperative thermal transition of α-hLA is apparently absent in this form. The first peak represents the unfolding transition from the native to an unfolding intermediate state (N–I transition) and the second peak represents that from the intermediate to the thermally unfolded state (I–U transition). We interpret that the cooperative thermal transition, which is observed between the intermediate and the thermally unfolded states of CML and EML, comes from the native-like packing interaction in their intermediate states. Furthermore, to examine the role of the stabilization mechanism of CML intermediate, we constructed four variant CMLs (H21G, I56L, A93S and V109K), in which the residues of CML are substituted for those of EML, and also investigated their thermal stability. Especially the His21 and Val109 of CML play a role in stabilization of the intermediate state and their contributions to the unfolding free energy are estimated to be 2.0 and 1.8 kJ/mol, respectively. From the results of the mutational analysis, a few differences in the local helical interactions within the α-domain are found to be predominant in stabilizing the intermediate state.
|Additional Information:||© 2001 Oxford University Press. Received April 19, 2001. Accepted September 4, 2001. Revision received August 2, 2001. We gratefully acknowledge the invaluable advice of Professor Isao Tanaka, Dr Min Yao of the Division of Biological Sciences, Graduate School of Science, Hokkaido University, Dr Mineyuki Mizuguchi of the Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University and of Dr Munehito Arai of The University of Tokyo. We also thank Ms Hiroko Yamamoto (Hokkaido University) for producing the variant CMLs. This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fishery of Japan and by Fellowships from the Japan Society for the Promotion of Science for Young Scientists (T.K.).|
|Subject Keywords:||canine milk lysozyme cooperativity differential scanning calorimetry thermodynamics three-state transition|
|Official Citation:||Takumi Koshiba, Yoshihiro Kobashigawa, Makoto Demura, and Katsutoshi Nitta Energetics of three-state unfolding of a protein: canine milk lysozyme Protein Eng. (2001) 14(12): 967-974 doi:10.1093/protein/14.12.967|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||21 Nov 2011 16:31|
|Last Modified:||21 Nov 2011 16:31|
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