Structure/function analysis of understudied pH-sensitive Kir4.2 channels in vitro with conformation specific nanobodies
Structure/function analysis of understudied pH-sensitive Kir4.2 channels in vitro with conformation specific nanobodies
批准号:
10217510
负责人:
Sun Joo Lee
金额:
$15.75万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2021
资助国家:
美国
项目状态:
已结题
起止时间:
2021-04-15 至 2022-08-14
关键词:
ArrhythmiaAtrial Heart Septal DefectsBiochemicalBiochemistryBiological AssayC-terminalCellsCholesterolComplexCongenital diaphragmatic herniaCryoelectron MicroscopyDefectDependenceDetergentsDevelopmentDiabetes MellitusDiseaseDockingDown SyndromeDrug ScreeningDrug or chemical Tissue DistributionElectrophysiology (science)EpilepsyEpitopesFamilyFunctional disorderGenerationsGenesGoalsHomeostasisHomoHumanImmunoglobulin GIn VitroIndividualInflammationIon ChannelLeadLibrariesLipidsLiposomesMembrane LipidsMembrane PotentialsMembrane ProteinsMethodsMolecularMolecular ConformationMutationNon-Insulin-Dependent Diabetes MellitusPathologicPatientsPharmaceutical PreparationsPhosphatidylinositol 4,5-DiphosphatePhysiologicalPolyunsaturated Fatty AcidsPositioning AttributePotassiumPropertyProtein ChemistryProteinsProtocols documentationProximal Renal Tubular AcidosisPublishingRegulationResolutionRestStructureSurfaceSystemTestingTherapeuticTimeTissuesTracerVascular DiseasesWorkX-Ray Crystallographybaseextracellulargene producthuman diseaseimprovedin silicoin vitro testinginsightinward rectifier potassium channellarge scale productionmutantnanobodiesnovelnovel strategiesnovel therapeuticsparticleprogramsprotein purificationreconstitutionscreeningsimulationsmall moleculestable cell linesystemic juvenile idiopathic arthritistherapeutic targettoolweb site
中文摘要
项目摘要
内向整流钾(Kir)通道调节许多组织的兴奋性,
Kir通道基因的突变导致多种疾病。的长期目标
该项目旨在了解未充分研究的pH敏感Kir 4的分子细节
子家庭频道以前,我定义了本体阴离子的分子机制,
Kir 2通道的脂质调节。这些先前的研究是由我的
Kir 2真核表达系统的建立及纯化方法的研究
通道,以及独特地结合多种方法(1)计算
对接模拟,(2)重组纯化蛋白的功能测定,
合成脂质体,和(3)高分辨率结构测定。这些研究
为获得对未充分研究的pH敏感Kir 4的新理解提供了模板
分子水平上的通道。我们将建立一个高效的表达系统
以及用于大规模纯化Kir4.2同源物和
Kir4.2/Kir5.1异源四聚体蛋白的研究不足,
对功能状态特异的纳米抗体,
在某些功能状态下的决定,但也有治疗潜力。
一旦确立,我们将利用同样的“三重结合”办法,
确定这些未充分研究的通道调节和功能的分子细节。
英文摘要
Project Summary
Inward rectifying potassium (Kir) channels regulate excitability in many tissues, and
multiple diseases result from mutations of Kir channel genes. The long-term goal of
this project is to understand the molecular details of understudied pH-sensitive Kir4
sub-family channels. Previously, I defined molecular mechanisms of bulk anionic
lipid regulation of Kir2 channels. These previous studies were made possible by my
establishing a new expression system and purification protocols for eukaryotic Kir2
channels, as well as uniquely combining the multiple methods of (1) computational
docking simulations, (2) functional assays of purified proteins reconstituted in
synthetic liposomes, and (3) high resolution structure determination. These studies
provide a template for gaining new understanding of understudied pH-sensitive Kir4
channels at the molecular level. We will establish an efficient expression system
and a purification protocol for large scale purifications of Kir4.2 homo- and
Kir4.2/Kir5.1 hetero-tetramer proteins that are understudied as well as develop
nanobodies specific to functional states that will not only facilitate structure
determinations in certain functional states but also have therapeutic potentials.
Once established, we will utilize the same `triple combination' of approaches to
determine molecular details of these understudied channel regulation and function.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文