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Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods

State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China
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Int. J. Mol. Sci. 2010, 11(7), 2584-2596; https://doi.org/10.3390/ijms11072584
Received: 21 May 2010 / Revised: 9 June 2010 / Accepted: 18 June 2010 / Published: 25 June 2010
(This article belongs to the Section Biochemistry)
The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK) thermal denaturation were studied by differential scanning calorimetry (DSC), CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK). The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities. View Full-Text
Keywords: differential scanning calorimetry; human brain-type creatine kinase; intrinsic fluorescence; stepwise transitions; thermal denaturation differential scanning calorimetry; human brain-type creatine kinase; intrinsic fluorescence; stepwise transitions; thermal denaturation
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Gao, Y.-S.; Su, J.-T.; Yan, Y.-B. Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods. Int. J. Mol. Sci. 2010, 11, 2584-2596.

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