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

• 批准号: 9322172
• 负责人: Riccardo Olcese
• 金额: $ 20.66万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/9322172
• 关键词: Address; Adopted; Agreement; Architecture; Bacteriophage T4; Biological Process; Cereals; Ciona intestinalis; Complex; Crystallization; Data; Dependence; Development; Dimensions; Disease; Dyes; Environment; Evaluation; Fluorescence Resonance Energy Transfer; Fluorometry; Gold; Health; High Pressure Liquid Chromatography; Integral Membrane Protein; Investigation; Ion Channel; Ion Channel Protein; Ions; Kinetics; Label; Length; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Movement; Muramidase; Muscle Contraction; Oocytes; Optical Methods; Optics; Peptides; Phosphoric Monoester Hydrolases; Physiological; Positioning Attribute; Probability; Process; Property; Proteins; Protocols documentation; Radial; Regulation; Resolution; Rest; Side; Signal Transduction; Site; Structural Models; Structural Protein; Structure; Synaptic Transmission; Temperature; Testing; Time; Toxin; Tryptophan; Vertebral column; Viral Proteins; Work; X-Ray Crystallography; density; experimental study; flexibility; fluorophore; human data; innovation; interest; large-conductance calcium-activated potassium channels; luminescence resonance energy transfer; member; molecular dynamics; multidisciplinary; nanometer; nanoscale; next generation; operation; polyproline; protein function; protein structure; protein structure function; retinal rods; structural biology; synthetic peptide; tetramethylrhodamine maleimide; voltage; voltage clamp

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.

项目概要 结构生物学的圣杯，即同时定量测定蛋白质的结构和 的功能，仍然很难实现。该应用程序涉及一种新的尖端技术的开发 光学方法可以实时解析亚纳米级距离和距离变化： 距离分辨电压钳荧光测定法 (drVCF)。 drVCF 结合了小型、光谱相同、 带有 Trp 诱导碰撞猝灭的可变长度的 Cys 连接荧光团。至关重要的是，荧光团范围 荧光团分子生成的径向概率密度函数 (pdf) 解释了灵活性 动力学（MD）模拟。 pdf用于同时拟合多个标签的光信号和 获得与蛋白质结构直接相关的高度受限的距离信息（来自色氨酸侧链 到标记的 Cys Cα 原子）。 drVCF 包含其他光学结构方法（FRET、 LRET等），例如广泛的适用性和生理相关的实验条件；但也各具特色 优点，例如（i）测量分子内距离和功能相关距离的能力 变化具有非常细的颗粒（初步评估中的测量误差<2 Å），实际上排除了 亚基间和分子间信号污染（<2.2 nm 范围）； (ii) 实际结构数据的获取 时间，允许同时跟踪结构和结构变化的动力学； (三) 有能力 从传导通道获取数据，无需大型蛋白质辅助物，例如毒素荧光团或 大荧光蛋白； (iv) 不依赖于荧光团偶极子方向。正如所有科学方法一样， drVCF 带有假设和限制。在本提案中，drVCF 的功能和限制将是 在已建立的结构生物学模型中针对三个具体目标进行了评估。目标 1：验证新光学器件 使用刚性棒状测量功能相关分子内蛋白质距离 (drVCF) 的方法 已知长度的肽。正如 Stryer 和 Haugland 校准 FRET 所做的那样，drVCF 精度将通过以下方式进行评估 测量刚性聚脯氨酸肽的长度。目标 2：在充分表征的可溶性蛋白质中验证 drVCF 已知结构。 drVCF 将用于测量 T4 溶菌酶的分子内距离，这是溶菌酶的金标准 结构生物学，评估该方法在蛋白质中的适用性及其准确性。目标 3：验证 具有已知静息/活性结构的电压敏感膜蛋白中的 drVCF。电压感测 电压敏感磷酸酶（Ci-VSP）的结构域最近在静息和活动状态下结晶。 drVCF将与切开的卵母细胞电压钳结合，测试其测量电压依赖性的能力 距离变化。这种方法是在经过非常令人鼓舞的初步实验后开发的。这 拟议的研究可能会揭示实际的陷阱和局限性，但随之而来的是纠正和纠正的机会 完善这种高度创新且具有潜在突破性的方法。