DRVCF, a new optical method for real-time, high resolution, intramolecular distance measurements in conducting ion channels

DRVCF,一种新的光学方法,用于传导离子通道中的实时、高分辨率、分子内距离测量

基本信息

  • 批准号:
    9322172
  • 负责人:
  • 金额:
    $ 20.66万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2017
  • 资助国家:
    美国
  • 起止时间:
    2017-03-15 至 2019-02-28
  • 项目状态:
    已结题

项目摘要

PROJECT SUMMARY The holy grail of structural biology, i.e., the simultaneous quantitative determination of a protein’s structure and function, remains very difficult to attain. This application pertains to the development of a new, cutting-edge optical approach that allows the resolution of sub-nanometer-scale distances and distance changes in real time: distance-resolving Voltage Clamp Fluorometry (drVCF). drVCF combines the use of small, spectrally-identical, Cys-attached fluorophores of variable length with Trp-induced collisional quenching. Crucially, fluorophore range and flexibility are accounted for by radial probability density functions (pdfs) generated by fluorophore molecular dynamics (MD) simulations. The pdfs are used to simultaneously fit the optical signals of multiple labels and obtain highly constrained distance information immediately relevant to protein structure (from the Trp side-chain to the labeled Cys Cα atom). drVCF encompasses the benefits of other optical structural approaches (FRET, LRET, etc.), such as wide applicability and physiologically-relevant experimental conditions; but also distinct advantages, such as (i) the ability to measure intramolecular distances and functionally-relevant distance changes with a very fine grain (<2 Å measurement error in a preliminary evaluation), practically excluding intersubunit and intermolecular signal contamination (<2.2 nm range); (ii) the acquisition of structural data in real time, allowing the simultaneous tracking of structure and the kinetics of structural change; (iii) the ability to acquire data from conducting channels without large protein adjuncts such as toxin-mounted fluorophores or large fluorescent proteins; (iv) no dependence on fluorophore dipole orientation. As all scientific approaches, drVCF carries assumptions and limitations. In this proposal, the capabilities and limitations of drVCF will be evaluated in established models of structural biology, over three Specific Aims. Aim 1: Validate a New Optical Approach to Measure Functionally-relevant Intramolecular Protein Distances (drVCF) Using Rigid Rod-like Peptides of Known Length. As Stryer and Haugland did to calibrate FRET, drVCF accuracy will be evaluated by measuring the length of rigid polyproline peptides. Aim 2: Validate drVCF in a Well Characterized Soluble Protein of Known Structure. drVCF will be used to measure intramolecular distances in T4 lysozyme, a gold standard in structural biology, to evaluate the applicability of this approach in proteins and its accuracy. Aim 3: Validate drVCF in a Voltage-sensitive Membrane Protein with Known Resting/Active Structures. The voltage-sensing domain of the voltage sensitive phosphatase (Ci-VSP) was recently crystallized in the Resting and Active states. drVCF will be combined with cut-open oocyte voltage clamp to test its ability to measure voltage-dependent distance changes. This approach was developed following highly-encouraging preliminary experiments. The proposed studies may reveal practical pitfalls and limitations, but with them also the opportunity to rectify and refine this highly innovative and potentially ground breaking approach.
项目摘要 结构生物学的圣杯,即,同时定量测定蛋白质的结构, 功能仍然很难实现。本申请涉及开发一种新的尖端 光学方法,允许亚纳米尺度的距离和距离变化的分辨率在真实的时间: 距离分辨电压钳荧光测定法(drVCF)。drVCF结合了使用小的,光谱相同的, 具有Trp诱导的碰撞淬灭的可变长度的Cys连接的荧光团。关键是,荧光团范围 和柔性由荧光团分子产生的径向概率密度函数(PDF)来解释 动力学(MD)模拟。PDF用于同时拟合多个标签的光学信号, 获得与蛋白质结构直接相关的高度约束的距离信息(从Trp侧链 标记的Cys C α原子)。drVCF包括其它光学结构方法(FRET, LRET等),例如广泛适用性和生理学相关的实验条件;而且还不同 优点,例如(i)测量分子内距离和功能相关距离的能力 以非常细的颗粒变化(初步评估中测量误差<2 μ m),实际上不包括 亚基间和分子间信号污染(<2.2 nm范围);(ii)采集真实的结构数据 时间,允许同时跟踪结构和结构变化的动力学;(iii)能够 - 从没有大蛋白质探针的传导通道获取数据,例如毒素固定的荧光团, 大的荧光蛋白;(iv)不依赖于荧光团偶极取向。正如所有的科学方法一样, drVCF带有假设和限制。在本提案中,drVCF的功能和局限性将 在已建立的结构生物学模型中进行评估,超过三个特定目标。目标1:新光学 利用刚性棒状探针测量功能相关蛋白质分子内距离(drVCF)的方法 已知长度的肽。正如Stryer和Haugland校准FRET所做的那样,drVCF准确度将通过以下方法进行评价: 测量刚性聚脯氨酸肽的长度。目的2:在充分表征的可溶性蛋白中的HoddrVCF 已知的结构。drVCF将用于测量T4溶菌酶中的分子内距离,T4溶菌酶是一种金标准, 结构生物学,以评估这种方法在蛋白质中的适用性及其准确性。目标3: drVCF在一个电压敏感的膜蛋白与已知的静止/活性结构。电压感测 电压敏感性磷酸酶(Ci-VSP)的结构域最近在静息和活性状态下结晶。 drVCF将与切开的卵母细胞电压钳结合,以测试其测量电压依赖性的能力。 距离变化。这种方法是在非常令人鼓舞的初步实验之后开发的。的 拟议的研究可能会揭示实际的陷阱和局限性,但也有机会纠正和 完善这种高度创新和潜在的突破性方法。

项目成果

期刊论文数量(0)
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Riccardo Olcese其他文献

Riccardo Olcese的其他文献

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{{ truncateString('Riccardo Olcese', 18)}}的其他基金

Sodium Dependent Inactivation of the Na+-Ca2+ exchange: Relevance to Cardiac Function
Na-Ca2 交换的钠依赖性失活:与心脏功能的相关性
  • 批准号:
    10531590
  • 财政年份:
    2020
  • 资助金额:
    $ 20.66万
  • 项目类别:
The Molecular Transitions that Initiate EC Coupling in Skeletal Muscle
骨骼肌中启动 EC 偶联的分子转变
  • 批准号:
    10594420
  • 财政年份:
    2019
  • 资助金额:
    $ 20.66万
  • 项目类别:
The Molecular Transitions that Initiate EC Coupling in Skeletal Muscle
骨骼肌中启动 EC 偶联的分子转变
  • 批准号:
    10371036
  • 财政年份:
    2019
  • 资助金额:
    $ 20.66万
  • 项目类别:
The Late L-type Ca Current as the Target for a New Class of Antiarrhythmics
晚期 L 型 Ca 电流作为新型抗心律失常药物的靶点
  • 批准号:
    9915944
  • 财政年份:
    2017
  • 资助金额:
    $ 20.66万
  • 项目类别:
Voltage-driven Structural Transitions in Voltage-Gated Calcium Channels
电压门控钙通道中电压驱动的结构转变
  • 批准号:
    9389512
  • 财政年份:
    2014
  • 资助金额:
    $ 20.66万
  • 项目类别:
Voltage-driven Structural Transitions in Voltage-Gated Calcium Channels
电压门控钙通道中电压驱动的结构转变
  • 批准号:
    9178075
  • 财政年份:
    2014
  • 资助金额:
    $ 20.66万
  • 项目类别:
Voltage-driven Structural Transitions in Voltage-Gated Calcium Channels
电压门控钙通道中电压驱动的结构转变
  • 批准号:
    9277214
  • 财政年份:
    2014
  • 资助金额:
    $ 20.66万
  • 项目类别:
BK(Ca) channel in heart mitochondria
心脏线粒体中的 BK(Ca) 通道
  • 批准号:
    8806591
  • 财政年份:
    2012
  • 资助金额:
    $ 20.66万
  • 项目类别:
Molecular Approaches to Arrhythmia Therapy
心律失常治疗的分子方法
  • 批准号:
    8852673
  • 财政年份:
    2005
  • 资助金额:
    $ 20.66万
  • 项目类别:
Molecular Approaches to Arrhythmia Therapy
心律失常治疗的分子方法
  • 批准号:
    8376291
  • 财政年份:
    2005
  • 资助金额:
    $ 20.66万
  • 项目类别:

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