Lakey J H, Duché D, González-Mañas J M, Baty D, Pattus F
European Molecular Biology Laboratory, Heidelberg, F.R.G.
J Mol Biol. 1993 Apr 5;230(3):1055-67. doi: 10.1006/jmbi.1993.1218.
The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this study fluorescence resonance energy transfer spectroscopy was used to determine the position of this helical hairpin in the membrane bound state. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulpho-1-naphthyl)ethylenediamine (I-AEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Five mutants I26C (helix 1), F105C (between helices 4 and 5), G166CJ (helix 8), A169C (helix 8-9), G176C (helix 9) were used. All mutants show wild-type binding activity to phosphatidylglycerol vesicles as judged by fluorescence polarization anisotropy, emission wavelength changes and brominated lipid quenching. The three tryptophan residues were used as a compound donor to AEDANS in resonance energy transfer distance determinations. The distances obtained for the soluble form were equal to those found in the crystal structure. On adding vesicles under conditions where intermolecular transfer was avoided the indicated distances increased; I26(10.9 A) F105(3.4 A), G166(3.3 A), A169(1.9 A) and G176(2.9 A). This confirms that, in the absence of a membrane potential, helices 1 and 2 open out onto the membrane surface whilst the helical hairpin remains closely packed against the rest of the structure. The insertion of this hairpin is thus not the driving force behind colicin membrane binding.
大肠杆菌素A的离子通道形成C末端片段与带负电荷的脂质囊泡结合，为可溶性蛋白质插入脂质双层提供了一个例子。已知其可溶性结构由包含疏水螺旋发夹的十螺旋束组成。在本研究中，荧光共振能量转移光谱用于确定该螺旋发夹在膜结合状态下的位置。将一种外在探针N'-(碘乙酰基)-N'-(5-磺基-1-萘基)乙二胺(I-AEDANS)连接到每个都带有独特半胱氨酸残基的突变蛋白上。使用了五个突变体I26C(螺旋1)、F105C(在螺旋4和5之间)、G166CJ(螺旋8)、A169C(螺旋8-9)、G176C(螺旋9)。通过荧光偏振各向异性、发射波长变化和溴化脂质猝灭判断，所有突变体对磷脂酰甘油囊泡均显示出野生型结合活性。在共振能量转移距离测定中，三个色氨酸残基用作AEDANS的复合供体。可溶性形式获得的距离与晶体结构中发现的距离相等。在避免分子间转移的条件下加入囊泡时，所示距离增加；I26(10.9埃)、F105(3.4埃)、G166(3.3埃)、A169(1.9埃)和G176(2.9埃)。这证实了，在没有膜电位的情况下，螺旋1和2伸向膜表面，而螺旋发夹则与结构的其余部分紧密堆积。因此，这个发夹的插入不是大肠杆菌素与膜结合的驱动力。
