Abstract
Bcl2 associated X (Bax) protein is an apoptotic member in the Bcl-2 protein family and playing a key role in regulating the apoptotic signaling. However, it remains unclear about the stability and unfolding of Bax protein. This study has reported a comprehensive investigation on the stability of Bax and many Bax variants in 0 and 6 M GdnHCl using ESR, CD, and Thermofluor spectroscopy methods. Nitroxide-based spin label (designated as R1 side chain) was used to probe the changes in local environment of protein with the applied chemical and thermal denaturation. ESR spectra were collected from various sites spanning over the nine α-helices of Bax at temperatures −23, 2, and 25 oC, providing information about how local environment of the respective nine helices is changed with the presence of GdnHCl at varying temperatures. Based on the observed site-specific ESR spectral changes, we found that Bax can be divided into two structural regions, of which respond differently to the presence of GdnHCl. In a solution containing 6 M GdnHCl, the N-terminal region (i.e., the first 88 residues from the N terminus, namely the helices from α1 to α3) was found to unfold largely because the corresponding spectra became similar and exhibited a highly mobile state, whereas the C-terminal region (covering from α4 to α9) of Bax was found to retain to some extent its local structures and remain unfolded. Some of the spectra from the C-terminal region even showed an enhanced immobilization of the R1 side chain either in 6 M GdnHCl or at high temperatures, supporting a view that the C-terminal region retains a well-defined tertiary structure against chemical and thermal denaturation. This finding was evidently supported by the results of CD spectroscopy. The far-UV CD spectra confirmed an appreciable amount of α-helical content of Bax in 0 M GdnHCl at high temperatures (90 oC). Most importantly, CD signal in the near-UV region was observed to be significant in magnitude at 2 oC and continuously increase with increasing temperature, suggesting that aromatic interactions are present within Bax structure and playing an important role in stabilizing Bax against the denaturing effect of increasing temperature. The importance of aromatic interactions within Bax structure was further confirmed by structural calculations to show that a total of 12 aromatic pairs were involved in aromatic interactions in the C-terminal region. Moreover, our Thermofluor assay showed that a point mutation in the interior surrounded by α4, α5, and α6 (particularly in the sequence from 99 to 117) in the C-terminal region would largely disrupt the stability of the whole Bax protein because the interior was spatially crowded with residues involved in aromatic-aromatic and cation-pi interactions in the C-terminal region. As such, we conclude that a molten globule state of Bax protein, which is composed of a coil-like denatured segment in the N-terminal region and a dry core in the C-terminal region, can exist as a stable monomer in 6 M GdnHCl at room temperatures or in 0 M GdnHCl at temperatures up to 90 oC (provided that the core sequence from 99 to 117 is not mutated). Because the aromatic interactions contribute significantly to the stabilization of the C-terminal region, Bax was found to largely unfold only at high temperatures in 6 M GdnHCl. Moreover, our results have evidently ruled out the “indirect interaction mechanism” that protein denaturation occurs because denaturants preferentially solvate hydrophobic residues, which in turn destabilize the native structure of a protein. The C-terminal region of Bax is a highly stable molten globule intermediate and is resistant to the denaturation by either heat or 6 M GdnHCl. Only by combining the effects of thermal and chemical denaturation could we unfold the molten globule intermediate of Bax and distinguish its stabilities.