Losonczi J A, Olejniczak E T, Betz S F, Harlan J E, Mack J, Fesik S W
Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, IL 60064, USA.
Biochemistry. 2000 Sep 12;39(36):11024-33. doi: 10.1021/bi000919v.
The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.
Bcl-2蛋白家族在程序性细胞死亡的调控中起着关键作用。这些蛋白发挥功能的一种假定机制涉及在膜中形成离子通道。作为在膜环境中对这些蛋白进行结构表征的第一步，我们研究了一种Bcl-x(L)突变蛋白掺入小的去污剂胶束时的结构。这种形式的Bcl-x(L)缺少螺旋1和螺旋2之间的环（残基49 - 88）以及假定的C端跨膜螺旋（残基214 - 237）。在临界胶束浓度（CMC）以下，Bcl-x(L)在与促凋亡蛋白结合的疏水凹槽中结合去污剂。然而，在CMC以上，Bcl-x(L)会发生显著的构象变化。我们使用核磁共振（NMR）方法表征了胶束结合形式的Bcl-x(L)的二级结构。与水溶液中的Bcl-x(L)一样，溶解在十二烷基磷酰胆碱（DPC）胶束中的蛋白质结构由几个被环隔开的α螺旋组成。然而，Bcl-x(L)在胶束中各个螺旋的长度和位置与在水溶液中不同。通过对蛋白质 - 去污剂核Overhauser效应（NOE）和有限蛋白酶解的分析，研究了Bcl-x(L)在胶束中的位置。此外，通过NMR弛豫测量研究了胶束结合形式的Bcl-x(L)的流动性。基于这些研究，提出了一个关于Bcl-x(L)在胶束中的结构、动力学和位置的模型。在这个模型中，Bcl-x(L)在胶束中具有松散堆积的动态结构，螺旋1和6以及可能的螺旋5部分埋入胶束的疏水内部。蛋白质的其他部分位于胶束表面附近或外部。
