利用電子自旋共振方法研究熱與化學變性作用對於 Bax 蛋白質之穩定性情形

An Assessment of Bax Protein Stability by Thermal and Chemical Denaturation Using Cw-ESR

詹淳惠(Chan, Chun Hui)

指導教授 ： 江昀緯

國立清華大學/理學院/化學系/碩士(2015年)

摘要

Bax是一種參與細胞凋亡過程的蛋白質,其中作用機制已經有完整的研究,但是Bax本身的穩定性和變性性質卻很少人探討,因此這篇論文會著重在探討Bax蛋白的穩定性和變性性質。本篇論文會以電子自旋共振光譜儀、旋光光譜儀和Thermofluor來探討Bax在熱和化學作用下的性質,期盼能從根本上了解Bax蛋白質 。

經由電子自旋共振光譜儀的分析,Bax可以分成兩區域：N-terminal和C-terminal。N-terminal是第1到88號氨基酸,此區域在化學作用下會完全失去結構,而剩下的C-terminal卻仍然保有部份結構。從序列的特性來看,百分之九十的含苯環氨基酸都分布在C-terminal,而這些含苯環氨基酸在距離夠近的情況下會產生交互作用,進而穩定蛋白質結構,這就是Bax在化學變性作用下還能保有部份結構的原因。

旋光光譜儀在熱變性作用下顯示Bax仍然存在二級結構和三級結構,這呼應了前面電子自旋共振光譜儀的論點。再者,Thermofluor利用螢光也量化不同突變點的熱穩定性,發現把含苯環氨基酸取代掉的位點（或是空間上靠近含苯環氨基酸的位點）會使蛋白質的熱穩定性下降特別顯著,因此可以證明含苯環氨基酸之間的交互作用是穩定蛋白質結構很重要的因素。

綜合以上, Bax因為有含苯環氨基酸之間的作用力,使得不管是在高溫或是高濃度化學變性劑的環境下都能保有部份結構。

關鍵字

蛋白質 ； 蛋白結構 ； 電子自旋共振

並列摘要

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.

並列關鍵字

Bax ； Protein structure ； Electron spin resonance

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