Thuduppathy Guruvasuthevan R, Hill R Blake
Department of Biology, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Protein Sci. 2006 Feb;15(2):248-57. doi: 10.1110/ps.051807706. Epub 2005 Dec 29.
Regulation of programmed cell death by Bcl-xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH-dependence similar to the translocation domain of diphtheria toxin, where an acid-induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl-xL deltaTM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near- and far-UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl-xL deltaTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-xL deltaTM (deltaG(o)) was determined to be 15.8 kcal x mol(-1). Surprisingly, under conditions that favor a membrane conformation (pH 4.9), deltaG(o) was 14.6 kcal x mol(-1). These results differ from those obtained with many other membrane-insertable proteins where acid-induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl-xL and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl-xL from intracellular proteases as it undergoes this conformational change essential for its activity.
Bcl-xL对程序性细胞死亡的调控依赖于其溶液构象和整合膜构象。从溶液构象到膜构象的转变在这种调控中也很重要。这种构象变化表现出与白喉毒素转位结构域相似的pH依赖性，在没有脂质体的情况下，酸诱导的熔球构象介导了从溶液构象到膜构象的转变。相比之下，在没有脂质体的情况下，通过近紫外和远紫外圆二色光谱偏振法观察到，酸化后Bcl-xL deltaTM没有明显的构象变化。此外，通过Bcl-xL deltaTM的异核核磁共振光谱法未观察到酸化后有明显的局部构象变化。在有利于溶液构象的条件下（pH 7.4），Bcl-xL deltaTM的折叠自由能（deltaG(o)）被确定为15.8 kcal x mol(-1)。令人惊讶的是，在有利于膜构象的条件下（pH 4.9），deltaG(o)为14.6 kcal x mol(-1)。这些结果与许多其他可插入膜的蛋白质不同，在那些蛋白质中酸诱导的不稳定是很重要的。因此，必须有其他因素来破坏Bcl-xL的溶液构象并有利于其在pH 4.9时的膜构象。这些因素可能包括带负电荷的膜的存在或跨膜的静电势。因此，对于同时采用溶液构象和膜构象的蛋白质，可能不需要一个强制性的熔球中间体。熔球中间体的缺失可能已经进化出来，以保护Bcl-xL在经历这种对其活性至关重要的构象变化时不被细胞内蛋白酶降解。
