pH-Triggered Membrane Insertion of Proteins

pH 触发的蛋白质膜插入

基本信息

  • 批准号:
    8714001
  • 负责人:
  • 金额:
    $ 33.67万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2004
  • 资助国家:
    美国
  • 起止时间:
    2004-08-01 至 2017-07-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): This project is focused on deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the diphtheria toxin T-domain (DTT), which is considered to be a paradigm for cell entry of other toxins (e.g., tetanus and botulinum) and has a potential for targeted delivery of anti-cancer therapies. The pH-triggered insertion of DTT will also reveal general physicochemical principles underlying membrane protein assembly and signalyng on membrane interfaces. This first competing renewal of the project will capitalize on our progress in identifying key intermediate states along the insertion pathway, in establishing the concept of conformational switching for DTT action and in developing new methodologies for structural, kinetic and thermodynamic characterization of membrane protein refolding/insertion. The innovation of this proposal resides in the unique way that molecular dynamics (MD) simulations and sophisticated spectroscopic experiments will be brought together in order to understand molecular mechanisms which will bring clarity to a complex field. MD simulations will be used for (a) building atomic models consistent with low resolution spectroscopic data, and (b) guiding the experimental design to further verify them. Site-specific labeling of single-cysteine mutants and a battery of spectroscopic approaches (including FCS, fluorescence lifetime quenching, FRET, stopped-flow kinetic measurements) will be utilized to test the interface-directed refolding/insertion hypothesis, which assigns a special role to the bilayer interfacial region in modulating transmembrane insertion by assisting the formation of key intermediate states, shifting the balance of electrostatic and hydrophobic interactions and altering protonation properties of titratable residues. The nature of the conformational switching resulting in refolding, insertion and translocation transitions of DTT will be established through mutagenesis of His, Asp and Glu residues, guided by Thermodynamic Integration calculations. Various DTT mutants will be used to ascertain whether protonation of histidines assists in the unfolding of the protein in solution and promotes formation of a previously identified insertion-competent intermediate on the membrane interface, through electrostatic interactions with anionic lipids, while protonation of acidic residues enables transmembrane insertion. To gain insights into the pH-triggered membrane action of DTT, thus establishing the general physicochemical principles of membrane-protein interactions, we will pursue the following goals: (1) determine molecular details of the structural organization of key intermediate and final inserted states; (2) determine the free energy profile of transitions along the insertion pathway and determine how the properties of the bilayer modulate structural, thermodynamic and kinetic parameters of the DTT insertion; and (3) identify key residues responsible for pH-triggered functional conformational switching.
描述(由申请人提供):该项目的重点是破译白喉毒素T结构域(DTT)的pH依赖性重折叠和膜插入的分子机制,这被认为是其他毒素进入细胞的范例(例如,破伤风和肉毒杆菌),并且具有抗癌疗法的靶向递送的潜力。pH触发的DTT插入也将揭示膜蛋白组装和膜界面信号转导的一般物理化学原理。该项目的第一个竞争性更新将利用我们在确定插入途径沿着的关键中间状态,建立DTT作用的构象转换概念和开发膜蛋白重折叠/插入的结构,动力学和热力学表征的新方法方面的进展。该提案的创新在于将分子动力学(MD)模拟和复杂的光谱实验结合在一起的独特方式,以了解分子机制,这将使复杂的领域变得清晰。MD模拟将用于(a)建立与低分辨率光谱数据一致的原子模型,以及(B)指导实验设计以进一步验证它们。单半胱氨酸突变体的位点特异性标记和一系列光谱方法(包括FCS,荧光寿命淬灭,FRET,停流动力学测量)将被用来测试界面导向的重折叠/插入假设,该假设通过帮助关键中间状态的形成,在调节跨膜插入中为双层界面区域分配了特殊的作用,改变静电和疏水相互作用的平衡并改变可滴定残基的质子化性质。通过热力学积分计算指导,通过His、Asp和Glu残基的诱变确定导致DTT重折叠、插入和易位转变的构象转换的性质。将使用各种DTT突变体来确定组氨酸的质子化是否有助于蛋白质在溶液中的解折叠,并通过与阴离子脂质的静电相互作用促进膜界面上先前确定的插入能力中间体的形成,而酸性残基的质子化使跨膜插入成为可能。为了深入了解DTT的pH触发的膜作用,从而建立膜-蛋白质相互作用的一般物理化学原理,我们将追求以下目标:(1)确定关键中间和最终插入状态的结构组织的分子细节;(2)确定沿着插入路径的跃迁的自由能分布并确定双层的性质如何调节结构,DTT插入的热力学和动力学参数;和(3)鉴定负责pH触发的功能构象转换的关键残基。

项目成果

期刊论文数量(36)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations.
  • DOI:
    10.1016/j.bpc.2010.12.001
  • 发表时间:
    2011-02
  • 期刊:
  • 影响因子:
    3.8
  • 作者:
    Kyrychenko, Alexander;Sevriukov, Igor Yu;Syzova, Zoya A.;Ladokhin, Alexey S.;Doroshenko, Andrey O.
  • 通讯作者:
    Doroshenko, Andrey O.
Comparison of membrane insertion pathways of the apoptotic regulator Bcl-xL and the diphtheria toxin translocation domain.
  • DOI:
    10.1021/bi400926k
  • 发表时间:
    2013-11-12
  • 期刊:
  • 影响因子:
    2.9
  • 作者:
    Vargas-Uribe, Mauricio;Rodnin, Mykola V.;Ladokhin, Alexey S.
  • 通讯作者:
    Ladokhin, Alexey S.
Validation of depth-dependent fluorescence quenching in membranes by molecular dynamics simulation of tryptophan octyl ester in POPC bilayer.
  • DOI:
    10.1021/jp310638f
  • 发表时间:
    2013-05-02
  • 期刊:
  • 影响因子:
    3.3
  • 作者:
    Kyrychenko, Alexander;Tobias, Douglas J.;Ladokhin, Alexey S.
  • 通讯作者:
    Ladokhin, Alexey S.
Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state.
  • DOI:
    10.1021/bi400249f
  • 发表时间:
    2013-05-21
  • 期刊:
  • 影响因子:
    2.9
  • 作者:
    Vargas-Uribe, Mauricio;Rodnin, Mykola V.;Kienker, Paul;Finkelstein, Alan;Ladokhin, Alexey S.
  • 通讯作者:
    Ladokhin, Alexey S.
Measuring membrane penetration with depth-dependent fluorescence quenching: distribution analysis is coming of age.
通过深度依赖性荧光猝灭测量膜渗透:分布分析即将成熟。
{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

Melanie J Cocco其他文献

Melanie J Cocco的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ truncateString('Melanie J Cocco', 18)}}的其他基金

Trapping membrane proteins with adjuvant-carrying amphipols for vaccine formulati
用携带佐剂的两性聚合物捕获膜蛋白用于疫苗配制
  • 批准号:
    8711230
  • 财政年份:
    2011
  • 资助金额:
    $ 33.67万
  • 项目类别:
Trapping membrane proteins with adjuvant-carrying amphipols for vaccine formulati
用携带佐剂的两性聚合物捕获膜蛋白用于疫苗配制
  • 批准号:
    8188329
  • 财政年份:
    2011
  • 资助金额:
    $ 33.67万
  • 项目类别:
Trapping membrane proteins with adjuvant-carrying amphipols for vaccine formulati
用携带佐剂的两性聚合物捕获膜蛋白用于疫苗配制
  • 批准号:
    8324510
  • 财政年份:
    2011
  • 资助金额:
    $ 33.67万
  • 项目类别:
Trapping membrane proteins with adjuvant-carrying amphipols for vaccine formulati
用携带佐剂的两性聚合物捕获膜蛋白用于疫苗配制
  • 批准号:
    8521069
  • 财政年份:
    2011
  • 资助金额:
    $ 33.67万
  • 项目类别:
pH-Triggered Membrane Insertion of Proteins
pH 触发的蛋白质膜插入
  • 批准号:
    8513343
  • 财政年份:
    2004
  • 资助金额:
    $ 33.67万
  • 项目类别:
pH-Triggered Membrane Insertion of Proteins
pH 触发的蛋白质膜插入
  • 批准号:
    8183855
  • 财政年份:
    2004
  • 资助金额:
    $ 33.67万
  • 项目类别:
pH-Triggered Membrane Insertion of Proteins
pH 触发的蛋白质膜插入
  • 批准号:
    8331449
  • 财政年份:
    2004
  • 资助金额:
    $ 33.67万
  • 项目类别:
STRUCTURE/FUNCTION OF PHOSPHOLAMBAN IN MEMBRANES
膜中磷苯班的结构/功能
  • 批准号:
    2796256
  • 财政年份:
    1998
  • 资助金额:
    $ 33.67万
  • 项目类别:
STRUCTURE/FUNCTION OF PHOSPHOLAMBAN IN MEMBRANES
膜中磷苯班的结构/功能
  • 批准号:
    2545322
  • 财政年份:
    1997
  • 资助金额:
    $ 33.67万
  • 项目类别:
STRUCTURE/FUNCTION OF PHOSPHOLAMBAN IN MEMBRANES
膜中磷苯班的结构/功能
  • 批准号:
    2006010
  • 财政年份:
    1997
  • 资助金额:
    $ 33.67万
  • 项目类别:

相似海外基金

The Development of Novel Inhibitors of BCL2 Gene Expression as Anticancer Therape
开发新型 BCL2 基因表达抑制剂作为抗癌疗法
  • 批准号:
    8642980
  • 财政年份:
    2014
  • 资助金额:
    $ 33.67万
  • 项目类别:
BCL2 GENE FAMILY, APOPTOSIS RESISTANCE AND BREAST CANCE
BCL2 基因家族、细胞凋亡抗性和乳腺癌
  • 批准号:
    2795904
  • 财政年份:
    1996
  • 资助金额:
    $ 33.67万
  • 项目类别:
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了