Liu Q R, Crozel V, Levinthal F, Slatin S, Finkelstein A, Levinthal C
Department of Biology, Columbia University, New York 10027.
Proteins. 1986 Nov;1(3):218-29. doi: 10.1002/prot.340010304.
Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.
用多种蛋白酶或溴化氰（CNBr）切割大肠杆菌素E1分子会产生COOH末端片段，这些片段在平面脂质双层膜中具有与完整大肠杆菌素相似的通道形成活性。通过CNBr切割野生型分子获得的最小通道形成片段由C末端的152个氨基酸组成。通过使用寡核苷酸定向诱变，我们沿着这152个氨基酸的肽段制作了9个突变体，其中一个氨基酸被甲硫氨酸取代，以创建一个新的CNBr切割位点。在平面双层膜中，具有通道形成活性的最小CNBr切割C末端片段是在新的甲硫氨酸位置428处切割产生的，有94个氨基酸，而在位置447处切割新的甲硫氨酸产生的75个氨基酸肽段没有通道活性。因此，大肠杆菌素E1通道形成结构域的NH2末端似乎位于残基428和447之间。然而，由于大肠杆菌素的最后六个C末端残基可以在不改变活性的情况下被去除，形成通道所需的氨基酸数量为88个或更少。此外，大肠杆菌素E1中独特的半胱氨酸残基被甘氨酸取代，然后沿着肽段在连续位置放置半胱氨酸制作了9个突变体，最终用作巯基连接位点以确定被取代氨基酸的局部环境。在制作21个突变体的过程中，8个带电荷的残基被不带电荷的甲硫氨酸或半胱氨酸取代，而完整分子的生物学活性没有改变。先前有人提出，大肠杆菌素E1通道的构象是由五个或六个α螺旋形成的桶状结构，每个α螺旋有20个氨基酸跨越膜，在膜边界处有两到四个残基形成转折。我们发现88个氨基酸可以形成一个活性通道，再结合最近报道的化学计量学证据表明该通道是单体，这排除了该模型，并增加了可用于构建通道分子模型的重要限制条件。
