Energetics of three-state unfolding of a protein: Canine milk lysozyme

Takumi Koshiba, Yoshihiro Kobashigawa, Makoto Demura, Katsutoshi Nitta

Research output: Contribution to journalArticle

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Abstract

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 Val09 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 k J/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.

Original languageEnglish
Pages (from-to)967-974
Number of pages8
JournalProtein Engineering
Volume14
Issue number12
Publication statusPublished - Dec 1 2001

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Protein Unfolding
Milk Proteins
Muramidase
Canidae
Enzymes
Milk
Hot Temperature
Horses
Calcium
Stabilization
Lactalbumin
Emitter coupled logic circuits
Differential Scanning Calorimetry
Thermodynamics
Free energy
Differential scanning calorimetry
Thermodynamic stability
Ions

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Koshiba, T., Kobashigawa, Y., Demura, M., & Nitta, K. (2001). Energetics of three-state unfolding of a protein: Canine milk lysozyme. Protein Engineering, 14(12), 967-974.

Energetics of three-state unfolding of a protein : Canine milk lysozyme. / Koshiba, Takumi; Kobashigawa, Yoshihiro; Demura, Makoto; Nitta, Katsutoshi.

In: Protein Engineering, Vol. 14, No. 12, 01.12.2001, p. 967-974.

Research output: Contribution to journalArticle

Koshiba, T, Kobashigawa, Y, Demura, M & Nitta, K 2001, 'Energetics of three-state unfolding of a protein: Canine milk lysozyme', Protein Engineering, vol. 14, no. 12, pp. 967-974.
Koshiba T, Kobashigawa Y, Demura M, Nitta K. Energetics of three-state unfolding of a protein: Canine milk lysozyme. Protein Engineering. 2001 Dec 1;14(12):967-974.
Koshiba, Takumi ; Kobashigawa, Yoshihiro ; Demura, Makoto ; Nitta, Katsutoshi. / Energetics of three-state unfolding of a protein : Canine milk lysozyme. In: Protein Engineering. 2001 ; Vol. 14, No. 12. pp. 967-974.
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