Equilibrium and Kinetics of the Folding and Unfolding of Canine Milk Lysozyme
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- 作者：Hiroyasu Nakatani ; Kosuke Maki ; Kimiko Saeki ; Tomoyasu Aizawa ; Makoto Demura ; Keiichi Kawano ; Shuji Tomoda ; Kunihiro Kuwajima
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 1, 2007
- 年：2007
- 卷：46
- 期：17
- 页码：5238 - 5251
- 全文大小：227K
- 年卷期：v.46,no.17(May 1, 2007)
- ISSN：1520-4995
文摘
The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptideand aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form ofthe protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populatedas an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from thekinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding withthe same molten globule intermediate. Although the observed kinetic refolding curves of both forms weresingle-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and theburst-phase intermediates of both forms were identical to each other with respect to their stability, indicatingthat the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the moltenglobule state observed at equilibrium. The 
-value analysis, based on the effect of Ca2+ on the foldingand unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transitionstate of folding. A comparison of the result with that previously reported for 
-lactalbumin indicated thatthe folding initiation site is different between canine milk lysozyme and -lactalbumin, and hence, thefolding pathways must be different between the two proteins. These results thus provide an example ofthe phenomenon wherein proteins that are very homologous to each other take different foldingpathways. It is also shown that the native state of the apo form is composed of at least two species thatinterconvert.
-value analysis, based on the effect of Ca2+ on the foldingand unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transitionstate of folding. A comparison of the result with that previously reported for -lactalbumin indicated thatthe folding initiation site is different between canine milk lysozyme and -lactalbumin, and hence, thefolding pathways must be different between the two proteins. These results thus provide an example ofthe phenomenon wherein proteins that are very homologous to each other take different foldingpathways. It is also shown that the native state of the apo form is composed of at least two species thatinterconvert.