Baumeister R, Müller G, Hecht B, Hillen W
Lehrstuhl für Mikrobiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Federal Republic of Germany.
Proteins. 1992 Oct;14(2):168-77. doi: 10.1002/prot.340140204.
The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.
源自Tn10的四环素阻遏物包含一段氨基酸序列，该序列与其他DNA结合蛋白的α-螺旋-转角-α-螺旋基序（HTH）具有高度同源性。HTH中五个最保守的氨基酸可能参与了该基序的结构形成。通过饱和诱变产生了95个四环素阻遏物的单氨基酸替代突变体，以此探究它们的功能作用。在体内定量测定了它们与四环素操纵基因的结合效率。所有功能突变体都含有与其在HTH中所推测作用相符的氨基酸替代。特别地，只有两个最小的氨基酸（丝氨酸、甘氨酸）能够替代推测的第一个α-螺旋中保守的丙氨酸而不丧失活性。第一个α-螺旋的最后一个位置、转角处的第二个位置以及第二个α-螺旋的第四个位置大多需要疏水残基。与野生型亮氨酸残基的谷氨酰胺替代突变体相比，第一个α-螺旋推测的C末端由一个活性更高的天冬酰胺所支持。如用功能性的赖氨酸和精氨酸替代缬氨酸所示，转角靠近蛋白质表面。转角处第一个位置的甘氨酸残基可以被任何氨基酸替代，产生的突变体至少具有残留的四环素操纵基因亲和力。本文给出了四环素阻遏物HTH的结构模型。
