Massotte D, Yamamoto M, Scianimanico S, Sorokine O, van Dorsselaer A, Nakatani Y, Ourisson G, Pattus F
European Molecular Biology Laboratory, Heidelberg, Germany.
Biochemistry. 1993 Dec 21;32(50):13787-94. doi: 10.1021/bi00213a006.
The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged 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 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.
大肠杆菌素A的离子通道形成热解片段（thA）与带负电荷的囊泡结合，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一个包含疏水螺旋发夹的10螺旋束组成。在本研究中，采用部分蛋白酶解和质谱法来确定thA片段在膜结合状态下对胰蛋白酶和α-胰凝乳蛋白酶蛋白水解攻击的可及位点。电喷雾质谱法是鉴定裂解产物的一种相当有效的方法，即使是对于部分纯化的肽混合物，且仅通过N端测序进行少量对照时也是如此。这项工作证实，肽链的大部分位于膜表面，甚至疏水发夹也未受到脂质双层的保护而免受蛋白水解降解。在没有膜电位的情况下，大肠杆菌素A膜结合形式中的疏水发夹似乎没有以跨膜方向固定。
