Name: site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels
Uploaded: 2016-07-04T15:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

我们非常感谢Chakrapani实验室对稿件批判性阅读和评论的现任和前任成员。 这项工作是由卫生部授予全国学院（1R01GM108921）和美国心脏协会（NCRP科学家发展赠款12SDG12070069）和SC的支持。

The authors have nothing to disclose.

EPR谱已被证明是在一个近乎完美的环境量化膜蛋白的构象变化无与伦比的结构方法。这种方法可以让我们一窥了在高分辨率结构遮蔽X射线晶体学和低温电子显微镜蛋白质动力学的分子机制。但是，要考虑这种做法可能会影响一般适用于其他系统，并要牢记沿途的潜在实验路障的技术限制，是非常重要的。我们讨论一些这些方面的下面一起排除故障推荐的策略。 其中较常见的...

在过去的十年，五聚配体门控离子通道的结构理解（pLGIC）增长的跨越式发展，由于家庭的几名成员的高分辨率结构的众人。导致在该领域的当前进展的关键因素包括，原核pLGIC渠道发现，真核生物膜蛋白表达1-3主要进展，4-6和巨大的突破，结构的测定方法。7这些结构上提供了一个明确的共识pLGIC的三维结构的整体保护。然而，这似乎落后背后两大领域是这些渠道制剂的功能特性和通道功能的机械描述。 选通的构象变化是复杂的，发生在沿着通道的长度的60埃的距离和这些转换由广泛调制膜脂。特别是，负脂质，胆固醇和磷脂已经显示调节pLGIC 8-11的功能。尽管通道功能，这些脂质成分的确切作用尚不清楚，门完整的分子机制，需要在他们自然环境研究这些通道。定点自旋标记（SDSL）和电子顺磁共振（EPR）谱是首选在复原系统研究蛋白质动力学的技术。 EPR谱不被分子大小的限制（这是NMR）或样品的光学特性（如为荧光光谱法），由此允许在天然脂质条件重构全长构建体的测量。该技术是非常敏感的，并具有相对低的样品的要求（在微微摩尔的范围）。这两个方面使该技术非常适合于大型研究膜蛋白难以超过毫克来表达数量。 利用与定点自旋标记组合EPR谱是由韦恩哈贝尔和同事开发，并已被改编为研究的范围内的蛋白质的类型。12-24 EPR数据的已被用于研究的二级结构，在该蛋白质改变构象，膜 - 插入深度，以及蛋白质 - 蛋白质/蛋白质 - 配体相互作用。 该方法涉及在由定点诱变感兴趣位置半胱氨酸取代。为确保位点特异性标记，就必须以替代天然半胱氨酸与另一个氨基酸（ 例如 ，丝氨酸）来创建一个半胱氨酸少模板。到目前为止，最流行的自旋标记是一个特定硫醇MTSL:（1-氧基-2,2,5,5-四甲基Δ3吡咯啉-3-甲基）methanethiosulfonate通过二硫键桥连接到蛋白质上。由于其高特异性，相对小的尺寸（小于色氨酸稍大），和灵活接头区域的相容性，这自旋标记已被证明具有优异的反应性，即使埋入半胱氨酸。此外，为了最大限度地提高反应性，该蛋白质的标记反应是在洗涤剂溶解的形式进行。通过尺寸排阻色谱的过量的游离自旋标记的分离后，将蛋白质重组到脂质体或限定的脂质组合物的双层模仿系统。在一般情况下，半胱氨酸诱变耐受良好的蛋白质的大部分地区，和自旋探头的相对小的尺寸会导致极小扰动的二级和三级结构。以确保该变形保留野生型功能，标记和重构通道可以通过膜片钳测量来研究。 然后将标记的官能蛋白进行光谱测量，其基本上提供三种主要类型的信息:由linesh 12,14,15,20,22,23,25-27自旋探针动力学猿分析;探头顺松弛剂无障碍;和距离的分布。27 EPR距离通过两种不同的方法进行测定。第一种是基于所述连续波（CW）技术，其中，从自旋标签之间偶极相互作用产生的频谱展宽（在8 - 20埃的距离范围内）。用于确定距离28,29的第二个是一个脉冲的EPR方法，其中，距离测量可以延长至70埃。30-34在双电子电子共振（鹿），在自旋回波振幅振荡被分析以确定的距离和距离的分布。这里自旋回波是在偶极相互作用的频率调制。总之，这些参数被用来确定蛋白质的拓扑结构，二级结构元件，和蛋白质的构象变化。

<table><tbody><tr><td>Site-Directed Mutagenesis and Cys mutations</td><td></td><td></td><td></td></tr><tr><td>10x PfuUltra HF reaction buffer</td><td>Agilent Technologies</td><td>600380-52</td><td></td></tr><tr><td>dNTPS</td><td>New England BioLabs Inc&amp;lrm;</td><td>N0447L</td><td>10 mM each dNTP</td></tr><tr><td>pfu Ultra DNA polymerase</td><td>Agilent Technologies</td><td>600380-51</td><td>2.5 U/ul</td></tr><tr><td>DPNI</td><td>New England BioLabs Inc&amp;lrm;</td><td>R0176S</td><td>20,000 U/ml</td></tr><tr><td>XL10 GOLD</td><td>Agilent Technologies</td><td>200314</td><td></td></tr><tr><td>SOC media</td><td>New England BioLabs Inc&amp;lrm;</td><td>B9020S</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher Scientfic</td><td>BP905</td><td></td></tr><tr><td>LB media</td><td>Invitrogen</td><td>127957084</td><td></td></tr><tr><td>Miniprep kit</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>C43 competent cells</td><td>Lucigen</td><td>60446</td><td></td></tr><tr><td>Expression and Purification</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td>Fisher Scientfic</td><td>D16</td><td></td></tr><tr><td>Tryptone</td><td>Fisher Bioreagents</td><td>BP1421-500</td><td></td></tr><tr><td>Yeast extract</td><td>Amresco</td><td>J850</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Bioreagents</td><td>BP229</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Amresco</td><td>0705</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Amresco</td><td>0781</td><td></td></tr><tr><td>IPTG (isopropyl-thio-&amp;beta;-galactoside)</td><td>Gold Biotechnology</td><td>I2481C25</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma Life Science</td><td>T1503</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>DNase I</td><td>Sigma Life Science</td><td>DN25</td><td></td></tr><tr><td>PMSF</td><td>Amresco</td><td>M145</td><td></td></tr><tr><td>Leupeptine</td><td>Amresco</td><td>J580</td><td></td></tr><tr><td>Pepstatin</td><td>Amresco</td><td>J583</td><td></td></tr><tr><td>DDM (n-Docecyl-&amp;beta;-D-Maltopyranoside)</td><td>Anatrace</td><td>D310S</td><td></td></tr><tr><td>Amylose resin</td><td>New England BioLabs Inc&amp;lrm;</td><td>E8021L</td><td></td></tr><tr><td>TCEP</td><td>Amresco</td><td>K831</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientfic</td><td>BP118</td><td></td></tr><tr><td>Maltose</td><td>Acros Organics</td><td>329915000</td><td></td></tr><tr><td>Superdex 200 GL</td><td>GE Healthcare</td><td>17-5175-01</td><td></td></tr><tr><td>Empty polypropylene Chromatography column</td><td>BioRad</td><td>731-1550</td><td></td></tr><tr><td>Site-Directed Spin Labeling</td><td></td><td></td><td></td></tr><tr><td>MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) Methanethiosulfonate</td><td>Toronto Reaserch chemicals Inc</td><td>O873900</td><td></td></tr><tr><td>(1-acetoxy-2,2,5,5-tetramethyl-&amp;Delta;3-pyrroline-3-methyl) methanethiosulfonate</td><td>Toronto Reaserch chemicals Inc</td><td>A167900</td><td></td></tr><tr><td>DMSO</td><td>J.T. Baker</td><td>9224-01</td><td></td></tr><tr><td>Reconstitution</td><td></td><td></td><td></td></tr><tr><td>Asolectin lipid</td><td>Avanti polar lipids Inc</td><td>541602C</td><td></td></tr><tr><td>Biobeads (Polystyrine beads)</td><td>Bio Rad</td><td>152-3920</td><td></td></tr><tr><td>Methanol</td><td>Fisher chemicals</td><td>A413</td><td></td></tr><tr><td>FRET</td><td></td><td></td><td></td></tr><tr><td>Fluorescein-maleimide</td><td>ThermoFisher Scientific</td><td>F-150</td><td></td></tr><tr><td>Tetramethylrhodamine-maleimide</td><td>ThermoFisher Scientific</td><td>T-6027</td><td></td></tr><tr><td>POPC</td><td>Avanti polar lipids Inc</td><td>850457C</td><td></td></tr><tr><td>POPG</td><td>Avanti polar lipids Inc</td><td>840457C</td><td></td></tr><tr><td>&lt;em&gt;E.coli&lt;/em&gt; polar lipid extract</td><td>Avanti polar lipids Inc</td><td>100600C</td><td></td></tr><tr><td>HEPES</td><td>Sigma Life Science</td><td>H3375</td><td></td></tr><tr><td>EPR measurement</td><td></td><td></td><td></td></tr><tr><td>TPX plastic capillaries</td><td>Bruker</td><td>ER221</td><td></td></tr><tr><td>EDDA (Ethylenediamine-N, N&#039;-diacetic acid)</td><td>Aldrich</td><td>158186</td><td></td></tr><tr><td>Ni(OH)&lt;sub&gt;2&lt;/sub&gt;</td><td>Aldrich</td><td>283622</td><td></td></tr></tbody></table>

site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

自旋标记GLIC突变体的生化特征 按照上述描述的方案通常产生GLIC-MBP融合蛋白在10〜 - 12毫克/升培养物。虽然这个值可以在不同的突变体而变化，特别是对于埋在蛋白质内的位置时，产率可能会显著损害。在这些情况下，培养体积可能需要扩大。融合的裂解构建由HRV3C蛋白酶进行O / N方便。通过运行在SDS PAGE凝胶上?...

Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

site-directed-spin-labeling-epr-spectroscopic-studies-pentameric

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

10.6K 次观看.  Case Western Reserve University. This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

视频: Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

科研

生物化学

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

用核磁共振 (nmr) 和微尺度热能 (mst) 测量球状和丝状蛋白的相互作用

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

生物工程

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

<meta charset="utf8"></head><body>通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

化学

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

使用单分子FRET可视化膜受体的构象动力学

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

摘要

探索更多视频

此视频中的章节

单分子水平的高精度FRET用于生物分子结构测定

光谱分析 TRPV1 的纯化与重构

用核磁共振 (nmr) 和微尺度热能 (mst) 测量球状和丝状蛋白的相互作用

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

实时测量核苷酸与完整功能性膜蛋白的结合

顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合

通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

通过固体 NMR 对天然膜中的蛋白质进行高分辨率研究

使用单分子FRET可视化膜受体的构象动力学

研究膜蛋白的构象动力学原位网站的荧光标记