Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating.
Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating.
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全长KCSA的分子结构：细胞质结构域在离子渗透和激活门口中的作用。
DOI：
10.1085/jgp.117.2.165
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发表时间：
2001-02
影响因子：
3.8
通讯作者：
Perozo, E
中科院分区：
文献类型：
--
作者：
Cortes, DM;Cuello, LG;Perozo, E
使用定点自旋标记方法和顺磁共振EPR光谱，已确定的原核钾通道KcsA的NH 2和COOH末端的分子结构。产生半胱氨酸突变体（残基5-24和121-160）并自旋标记，并且在室温下从脂质体重构通道获得X带CW EPR谱。探针迁移率（ΔHo−1）、可及性参数（Δ O2和Δ NiEdda）和亚基间自旋-自旋相互作用（Ω）的数据被用作结构约束，以从一组模拟退火和约束分子动力学运行中构建这些胞质结构域的三维折叠模型。生成32个骨架结构并使用四重对称性进行平均，并且从8个最低能量运行获得最终平均结构。基于目前的数据，连同来自KcsA晶体结构的信息，构建了全长KcsA的三维折叠的模型。在该模型中，KcsA的NH 2末端形成锚定在膜-水界面处的α-螺旋，而COOH末端形成向细胞质延伸约40-50 μ m的右旋四螺旋束。COOH-末端缺失构建体的功能分析表明，虽然COOH末端在确定离子渗透特性中不起实质性作用，但它在pH依赖性门控机制中发挥调节作用。
The molecular architecture of the NH2 and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy. Cysteine mutants were generated (residues 5–24 and 121–160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (ΔHo−1), accessibility parameters (ΠO2 and ΠNiEdda), and inter-subunit spin-spin interaction (Ω) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH2 terminus of KcsA forms an α-helix anchored at the membrane–water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40–50 Å towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
2.9
作者：
Cortes, DM;Perozo, E
通讯作者：
Perozo, E
影响因子：
2.9
作者：
Gross, A;Columbus, L;Hubbell, WL
通讯作者：
Hubbell, WL
影响因子：
2.9
作者：
Heginbotham, L;Odessey, E;Miller, C
通讯作者：
Miller, C
DOI：
10.1085/jgp.114.4.551
发表时间：
1999-10
期刊：
The Journal of general physiology
影响因子：
--
作者：
Heginbotham L;LeMasurier M;Kolmakova-Partensky L;Miller C
通讯作者：
Miller C