绿脓杆菌apoazurin在溶液中存在两种天然构象(英文)

Two Conformations of Native Pseudomonas aeruginosa Apoazurin in Solution

如何解释绿脓杆菌apoazurin变性过程的复杂机制是一个有争议的问题.最近的研究表明apoazurin的复杂变性机制可以归结为其天然态存在着至少两种构象.利用内源荧光发射谱和圆二色谱进一步研究了apoazurin的脲变性机制,发现其稳态脲变性符合表观的二态过程,但其动力学为双相过程.在高浓度脲中快反应在几秒钟内完成,而慢反应要经过几个小时.快反应和慢反应的mu值分别为2.24和2.45kJ·mol-1·M-1,去折叠活化能的差值为22kJ·mol-1.时间分辨的荧光发射谱和圆二色谱可以用天然态和完全变性态的谱图通过一个固定的比例参数进行重建.结果表明,过去被广泛接受的存在着变性中间体的机制是不正确的,而apoazurin在天然态存在至少两种构象的假设是合理的.

The mechanism of complex unfolding process of Pseudomonas aeruginosa apoazurin is an arguing problem. Recent published results indicated that this problem could be resolved by hypothesizing two native conformations coexisting in solution. Urea-induced unfolding of apoazurin was investigated further using intrinsinc fluorescence and CD spectra. Equilibrium unfolding curves in urea could be depicted with an apparent two-state transition, but a biphase kinetic process. The fast unfolding process is finished within a few seconds as monitored by stopped-flow fluorescence intensity, whereas the slow process requires several hours for unfolding at high concentration of urea at room temperature. The mu values for the fast and slow phases are 2.24 and 2.45 kJ·mol^-1·M^-1 respectively. The difference between their unfolding activation energy is 22 kJ·mol^-1. The time-resolved fluorescence emission spectra as well as the circular dichroism spectra just after manual mixing of protein and denaturant could be simulated by superposition of the spectra of the native and fully unfolded protein with the same coefficient of 0.37. This strongly suggests the three-state mechanism with a partially unfolded intermediate on its pathway is inadequate. The hypothesis of two native conformations coexistence is a reasonable selection.