Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme

Takumi Koshiba, Min Yao, Yoshihiro Kobashigawa, Makoto Demura, Atsushi Nakagawa, Isao Tanaka, Kunihiro Kuwajima, Katsutoshi Nitta

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-α-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 Å to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 °C higher than that of equine milk lysozyme. Furthermore, the 1H NMR spectra of canine milk lysozyme at 60 °C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 °C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.

Original languageEnglish
Pages (from-to)3248-3257
Number of pages10
JournalBiochemistry
Volume39
Issue number12
DOIs
Publication statusPublished - Mar 28 2000

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Muramidase
Thermodynamics
Canidae
Molten materials
Milk
R388
Lactalbumin
Protein Unfolding
Calorimetry
Proteins
Temperature
Protein Stability
X ray crystallography
X Ray Crystallography
Freezing
Nuclear magnetic resonance spectroscopy
Horses
Melting point
Protons
Spectrum Analysis

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Koshiba, T., Yao, M., Kobashigawa, Y., Demura, M., Nakagawa, A., Tanaka, I., ... Nitta, K. (2000). Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme. Biochemistry, 39(12), 3248-3257. https://doi.org/10.1021/bi991525a

Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme. / Koshiba, Takumi; Yao, Min; Kobashigawa, Yoshihiro; Demura, Makoto; Nakagawa, Atsushi; Tanaka, Isao; Kuwajima, Kunihiro; Nitta, Katsutoshi.

In: Biochemistry, Vol. 39, No. 12, 28.03.2000, p. 3248-3257.

Research output: Contribution to journalArticle

Koshiba, T, Yao, M, Kobashigawa, Y, Demura, M, Nakagawa, A, Tanaka, I, Kuwajima, K & Nitta, K 2000, 'Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme', Biochemistry, vol. 39, no. 12, pp. 3248-3257. https://doi.org/10.1021/bi991525a
Koshiba T, Yao M, Kobashigawa Y, Demura M, Nakagawa A, Tanaka I et al. Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme. Biochemistry. 2000 Mar 28;39(12):3248-3257. https://doi.org/10.1021/bi991525a
Koshiba, Takumi ; Yao, Min ; Kobashigawa, Yoshihiro ; Demura, Makoto ; Nakagawa, Atsushi ; Tanaka, Isao ; Kuwajima, Kunihiro ; Nitta, Katsutoshi. / Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme. In: Biochemistry. 2000 ; Vol. 39, No. 12. pp. 3248-3257.
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