Sequential events in the irreversible thermal denaturation of human brain-type creatine kinase by spectroscopic methods

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.

Keywords: differential scanning calorimetry; human brain-type creatine kinase; intrinsic fluorescence; stepwise transitions; thermal denaturation.

Figure 1.

Structure of HBCK monomer (PDB ID: 3DRE) [27]. N and C denote the N- and C-terminus of the protein, respectively. The positions of the four Trp residues are highlighted by the space-filling model.

Figure 2.

Temperature dependence of the heat flow of hBBCK thermal denaturation monitored by DSC. The heating rate was 1 K/min. The protein concentration was 1 mg/mL. The first scan is shown by the solid line, and the dotted line represents the second scan, which was performed by reheating the samples after cooling from the first scan. The negative peaks in the DSC profile are endothermic.

Figure 3.

Intrinsic fluorescence spectra of the thermally inactivated and reactivated hBBCK samples. The inactivated samples were prepared by heating the enzyme solutions (0.2 mg/mL) at 25 °C, 50 °C, and 60 °C for 10 min, while the reactivated samples were prepared by incubating the inactivated samples on ice for 24 h. The intrinsic fluorescence was measured with an excitation wavelength of 280 nm.

Figure 4.

hBBCK thermal denaturation monitored by CD spectroscopy. (A) Typical CD spectra of hBBCK recorded at a given temperature after 2 min equilibration. (B) Changes in the ellipticity at 222 nm with the increase of temperature. The CD data are presented as mean residue molar ellipticity ([θ]_MRW).

Figure 5.

hBBCK thermal denaturation monitored by intrinsic fluorescence spectroscopy excited at 295 nm. (A) Fluorescence spectra of hBBCK recorded at the given temperature after 2 min equilibration. (B) Dependence of I₃₂₀/I₃₆₅ on temperature.

Figure 6.

Changes in the oligomeric states during hBBCK thermal denaturation monitored by resonance light scattering.

Figure 7.

Phase diagram analysis of the fluorescence data shown in Figure 5. The diagram was constructed by plotting I₃₂₀ versus I₃₆₅. The I₃₂₀ and I₃₆₅ values were normalized by the maximum value in each set of the fluorescence data.

Figure 8.

A summary of the transition curves obtained by various probes. The activity data were from [43].