Abstract
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.
Highlights
When denatured by chemical denaturants, the unfolding of small single-domain proteins is generally a “all” or “none” two-state process, while that of large multimeric or multi-domain proteins is usually a much more complex process that involves a hierarchy of structural transitions [1,2]
Our previous study has indicated that the thermal inactivation of human BBCK (hBBCK) is partially reversible at temperatures below 55 °C, but no activity could be recovered by 24 h reactivation on ice for samples pre-heated at temperatures above 60 °C [43]
The sequential events in hBBCK thermal denaturation were studied by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy
Summary
When denatured by chemical denaturants, the unfolding of small single-domain proteins is generally a “all” or “none” two-state process, while that of large multimeric or multi-domain proteins is usually a much more complex process that involves a hierarchy of structural transitions [1,2]. The folding mechanism of MMCK, when denatured or renatured in guanidine chloride or urea solutions, has been characterized as a multi-state process involving several intermediates [29,30,31,32,33,34,35,36,37]. Both kinetic and equilibrium folding studies have revealed the existence of a monomeric intermediate with unfolded NTD and folded CTD, and a dimeric inactive intermediate maintaining most of the native secondary structures. The sequential events in hBBCK thermal denaturation were investigated by spectroscopic methods
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