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
- 来源:
- 关键词:AddressAdoptedAgreementArchitectureBacteriophage T4Biological ProcessCerealsCiona intestinalisComplexCrystallizationDataDependenceDevelopmentDimensionsDiseaseDyesEnvironmentEvaluationFluorescence Resonance Energy TransferFluorometryGoldHealthHigh Pressure Liquid ChromatographyIntegral Membrane ProteinInvestigationIon ChannelIon Channel ProteinIonsKineticsLabelLengthMeasurementMeasuresMediatingMembraneMembrane ProteinsMolecularMolecular ConformationMovementMuramidaseMuscle ContractionOocytesOptical MethodsOpticsPeptidesPhosphoric Monoester HydrolasesPhysiologicalPositioning AttributeProbabilityProcessPropertyProteinsProtocols documentationRadialRegulationResolutionRestSideSignal TransductionSiteStructural ModelsStructural ProteinStructureSynaptic TransmissionTemperatureTestingTimeToxinTryptophanVertebral columnViral ProteinsWorkX-Ray Crystallographydensityexperimental studyflexibilityfluorophorehuman datainnovationinterestlarge-conductance calcium-activated potassium channelsluminescence resonance energy transfermembermolecular dynamicsmultidisciplinarynanometernanoscalenext generationoperationpolyprolineprotein functionprotein structureprotein structure functionretinal rodsstructural biologysynthetic peptidetetramethylrhodamine maleimidevoltagevoltage clamp
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.
项目总结
结构生物学的圣杯,即同时定量测定蛋白质的结构和
功能,仍然很难实现。这项申请属于开发一种新的、尖端的
光学方法,允许实时分辨率亚纳米级的距离和距离变化:
距离分辨电压钳荧光法(DrVCF)。DrVCF结合了小的、光谱相同的、
Trp诱导的碰撞猝灭的可变长度的CyS连接的荧光团。至关重要的是,荧光团的范围
和灵活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用于同时适配多个标签的光信号
获得与蛋白质结构直接相关的高度受限的距离信息(从Trp侧链
标记的CysCα原子)。DRVCF包含其他光学结构方法的优点(FRET,
LRET等),如广泛的适用性和与生理相关的实验条件;但也有独特的
优势,例如(I)测量分子内距离和功能相关距离的能力
变化非常细小(初步评估中的测量误差),实际上排除了
亚基间和分子间信号污染(<;2.2 nm范围);(Ii)真实结构数据的获取
时间,允许同时跟踪结构和结构变化的动力学;(3)能够
从没有大的蛋白质附件的传导通道获取数据,例如毒素安装的荧光团或
大的荧光蛋白;(Iv)不依赖于荧光团的偶极取向。随着所有科学方法的临近,
DrVCF带有假设和限制。在本提案中,drVCF的功能和限制如下
在已建立的结构生物学模型中进行评估,超过三个具体目标。目标1:验证新的光纤
利用刚性棒状蛋白质测量功能相关分子内蛋白质距离(DrVCF)的方法
已知长度的多肽。就像Stryer和Hauland校准FRET一样,drVCF的准确性将通过以下方式进行评估
测量刚性多脯氨酸多肽的长度。目的2:验证一种特性良好的可溶性蛋白中的drVCF
已知结构的。DrVCF将用于测量T4溶菌酶的分子内距离,T4溶菌酶是
结构生物学,以评估这种方法在蛋白质中的适用性和准确性。目标3:验证
DrVCF是一种电压敏感膜蛋白,具有已知的静息/活性结构。电压传感技术
电压敏感型磷酸酶(Ci-VSP)的结构域最近在静息状态和活性状态下结晶。
DrVCF将与切开的卵母细胞电压钳相结合,以测试其测量电压依赖的能力
距离变了。这种方法是在非常令人鼓舞的初步实验之后开发出来的。这个
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
完善这一极具创新性和潜在开创性的方法。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(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
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Voltage-driven Structural Transitions in Voltage-Gated Calcium Channels
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