Stepwise selection of TetR variants recognizing tet operator 6C with high affinity and specificity
Stepwise selection of TetR variants recognizing tet operator 6C with high affinity and specificity
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DOI：
10.1006/jmbi.1997.1539
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发表时间：
1998-02-20
影响因子：
5.6
通讯作者：
Hillen, W
中科院分区：
文献类型：
--
作者：
Helbl, V;Tiebel, B;Hillen, W
TetR 识别 α 螺旋的第六个位置上的 Trp43 交换为 Arg，导致对 tetO-6% 产生新的 DNA 识别特异性，然而，它仅以低亲和力结合。通过识别 α 螺旋和转角最后位置的额外氨基酸交换，该突变体的特异性和亲和力显着增加，这很可能在 TetR-tetO 复合物的形成中发挥结构作用。 α-螺旋-转角-α-螺旋基序的最后一个残基是与其他 tetO 变体结合的鉴别器，并有效地有助于新识别的 tetO-6C 序列的亲和力。当与识别 α 螺旋中的残基结合时，该位置的短残基可提高序列特异性结合，从而直接读出识别的 tetO 序列。我们假设转角末端的小残基允许识别 α 螺旋在基序内占据最佳位置，以对 DNA 靶标进行计时。因此，允许与新识别的DNA直接和有利接触的残基不足以增加结合特异性和亲和力，而是需要伴随允许形成这些接触的额外交换。 (C) 1998 学术出版社有限公司。
The exchange of Trp43 to Arg in the sixth position of the TetR recognition alpha-helix leads to a new DNA recognition specificity for tetO-6%, however, it is bound with only low affinity. Specificity and affinity of this mutant were substantially increased by additional amino acid exchanges in the last positions of the recognition alpha-helix and the turn, which most likely play structural roles in the formation of the TetR-tetO complex. The last residue in the turn of the alpha-helix-turn-alpha-helix motif is a discriminator of binding to other tetO variants and contributes efficiently to the affinity for the newly recognized tetO-6C sequence. Short residues at this position improve sequence specific binding when combined with a residue in the recognition alpha-helix, which directly reads out the recognized tetO sequence. We assume that small residues at the end of the turn permit the recognition alpha-helix to assume the optimal position within the motif for clocking to the DNA target. Thus, residues allowing direct and favourable contacts to the newly recognized DNA are not sufficient to increase the binding specificity and affinity, but need to be accompanied by additional exchanges allowing the formation of these contacts. (C) 1998 Academic Press Limited.