【作者】 岑成；

【导师】 郭万林；

【作者基本信息】 南京航空航天大学 ， 力学， 2009， 硕士

【摘要】 1998年KcsA晶体结构的发现,是从原子尺度所进行的对离子通道选择性研究的一次重大突破。之后,越来越多的注意力开始向离子通道的门控特性聚拢。然后,由于缺少单一蛋白在闭式和开式状态下的双晶体结构,通道门控方面的很多特性还不为人知。2006年和2009年先后获得的NaK通道闭式和开式结构,为研究离子通道门控特性提供了一个绝佳的机会。在此次硕士学位论文的工作中,我通过拉伸分子动力学(TMD)模拟了NaK通道由闭合态向开启态的转变过程,有两个重要发现。一个是提出了GIG的新铰链模式,并由此解释了旋转和弯曲运动的分离机制。之前的门控模型,要么认为旋转和弯曲单独发生,要么认为旋转弯曲在铰链以下均匀分布,我发现,在NaK通道门控过程中,内螺旋的旋转和弯曲运动的起始位置不同,前者集中于包含G87号铰链的GIG残基上;后者则自V91开始,分布在V91号残基以下的内螺旋区域。另一方面,旋转和弯曲沿螺旋的变化规律不同,前者在GIG区域即由0快速增加到最大值,而在GIG铰链域以下没有发生变化而始终维持在最大值;弯曲从V91开始沿内螺旋均匀增加,直至螺旋末端。旋转和弯曲分离机制的提出,大大丰富了我们对于离子通道门控特性的认识。二个是分析了NaK通道变形过程中,亚基间所围成的导水支道结构的变迁,以及它的堵塞机制。变迁方面,发现支道口径的变化是由内螺旋旋转运动所导致的,控制位点是V91,这和“门”的变迁机制不同,门的变迁是由内螺旋的旋转和弯曲运动共同导致的,从而明确了NaK通道两种导水方式的区别和联系,阐释了这两者与蛋白变形的关系。堵塞机制方面,发现内螺旋上苯丙氨酸F94侧链苯环和外螺旋F28侧链苯环形成的稳定构型,导致了支道外侧的堵塞,新发现的这个亚基内相互作用对于维持NaK开式构型的稳定也非常重要;发现内螺旋上F92在脱离相邻亚基疏水层对它的束缚后,发生了侧链苯环的不规则偏转,导致了支道内侧的随机性堵塞。更多还原

【Abstract】 The report of the crystal structure of KcsA in 1998 contributes to the breakthrough in revealing the atomic scale mechanisms of selectivity of potassium channels, after that, more and more attention was drawn to their gating properties. However, due to a lack of single channel crystal structures in both closed and open conformations, many dynamic properties concerning channel gating remain unclear. It provides a good opportunity to study potassium channel gating owing to the determination of the NaK channel structure of closed and open states in 2006 and 2009, respectively. In this thesis, by performing a targeted MD simulation stretching the closed structure of the NaK channel towards its open structure, I have two important findings.Firstly, we proposed a new GIG hinge model and use this model to explain the decoupling mechanism of twist and bending motions. Previous models either support bend opening, twist opening or regular bend/twist opening, whereas I find in the NaK channel gating that twist and bending motions starts in different sites of the inner helix, twist motions concentrate in the GIG motif; bending motions starts in V91 beneath the hinge. On the other hand, twist and bending motions changes differently along the inner helix, twist starts quickly in the GIG motif and changes to its maximum degree, but remains unchanged beneath the hinge; while bending increases linearly along the length of the inner helix until the C-terminal. This decoupling mechanism between bending and twist motions greatly contributes to our understanding of gating mechanisms of ion channels.Secondly, we analyzed the changes of the water permeating orifice and its blocking mechanisms during conformational changes of the NaK channel. The change of the diameter of the orifice is due to twisting motion in the inner helix and is constricted at residue V91. This mechanism differs from that of gate opening, which is contributed by both bending and twist motions in the inner helix. This finding specifies the differences and correlations between two“gates”of water permeation and explains their relations with conformational changes in the inner helix. About the blockage of the the orifice, the side chain benzene ring of F92 in the inner helix and F28 in the outer helix forms very stable interactions, which cause the blockage of the outer mouth of the orifice, this interaction is also very important for channel stabilization during its open state; Lacking the constriction from inter-subunit Hydropobic Patch, the benzene ring in the side chain of F92 swings irrugularly and causes random blockage of the inner mouth of the orifice.更多还原