STRUCTURAL STUDIES OF KCSA MUTANTS AND CHIMERAS

KCSA 突变体和嵌合体的结构研究

• 批准号: 8363398
• 负责人: Crina M Nimigean
• 金额: $ 0.31万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363398
• 关键词: Amino Acids; Binding; Binding Sites; Charge; Chemicals; Chimera organism; Complex; Coupling; Crystallography; Dependence; Diffuse; Discrimination; Funding; Grant; Hydrogen Bonding; Individual; Ions; Light; Mutation; National Center for Research Resources; Organism; Potassium Channel; Principal Investigator; Proteins; Protons; Research; Research Infrastructure; Resources; Site; Sodium Chloride; Source; Structure; Synchrotrons; United States National Institutes of Health; X-Ray Crystallography; cost; ionization; mutant; prevent

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Project 1 -- The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge renders the channel open up to pH 9.0 (Thompson et al. 2008). We proposed that these residues function as the proton-binding sites. At neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing, stabilizing the closed state. At acidic pH, these residues change their ionization state, thereby disrupting this network, favoring channel opening. Individual mutations of residues in this region result in modest shifts in the pH dependence of channel opening. Pair-wise mutation of certain amino acids in this region causes a marked shift in the pH dependence of channel opening suggesting significant energetic coupling between these residues. We hypothesize that the crystal structures of these mutants will shed light on the structural changes that occur at the inner helical bundle crossing upon channel gating. Project 2 -- Potassium channels allow K(+) ions to diffuse through their pores while preventing smaller Na(+) ions from permeating. Discrimination between these similar, abundant ions enables these proteins to control electrical and chemical activity in all organisms. Selection occurs at the narrow selectivity filter containing structurally identified K(+) binding sites. Selectivity is thought to arise because smaller ions such as Na(+) do not bind to these K(+) sites in a thermodynamically favorable way. We wish to examine how intracellular Na(+) and Li(+) interact with the KcsA pore and the permeant ions using X-ray crystallography.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 项目1 -细菌钾通道KcsA由高浓度的细胞内质子门控，允许通道在pH < 5.5时打开。用模拟其质子化对应物的残基取代KcsA N和C末端的关键可电离残基，使通道在pH 9.0时开放（Thompson et al. 2008）。我们认为这些残基起着质子结合位点的作用。在中性pH下，它们形成一个复杂的网络间和intrasubunit盐桥和氢键附近的束交叉，稳定的封闭状态。在酸性pH值下，这些残基改变其电离状态，从而破坏该网络，有利于通道开放。该区域中残基的单个突变导致通道开放的pH依赖性的适度变化。在这个区域中的某些氨基酸的成对突变导致通道开放的pH依赖性的显著转变，这表明这些残基之间的显著能量耦合。我们推测，这些突变体的晶体结构将揭示通道门控时发生在内部螺旋束交叉的结构变化。 项目2 --钾通道允许K（+）离子通过其孔隙扩散，同时阻止较小的Na（+）离子渗透。这些相似的、丰富的离子之间的区别使这些蛋白质能够控制所有生物体中的电和化学活性。选择发生在包含结构上鉴定的K（+）结合位点的窄选择性过滤器处。选择性被认为是因为较小的离子如Na（+）不能以化学上有利的方式结合到这些K（+）位点。我们希望使用X射线晶体学检查细胞内Na（+）和Li（+）如何与KcsA孔和渗透离子相互作用。