Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
FEBS Lett. 2010 Mar 19;584(6):1133-8. doi: 10.1016/j.febslet.2010.02.015. Epub 2010 Feb 12.
The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117-122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening.
负责 KcsA 质子敏感性的结构的分子性质已被确定为围绕内部螺旋束门的电荷簇。在这里，我们表明可以修饰这种质子传感器来构建 KcsA 的组成型开放形式，使其易于进行功能、光谱和结构分析。通过中和簇中位置 25、117-122 和 124 处所有酸性和碱性残基的电荷（但不是 E118），生成了一种突变的 KcsA，该突变体显示出直到 pH 9 的组成型开放通道活性。该突变体的结构表明，由于激活门似乎仅处于打开的早期阶段，因此完全打开可能受到晶格力的抑制。
