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

私たちは、原稿上の重要な読書とコメントChakrapaniラボの現在および過去のメンバーに非常に感謝しています。 この作品は、国立衛生研究所の助成金（1R01GM108921）とアメリカ心臓協会（NCRPサイエンティスト開発グラント12SDG12070069）によって、およびSCにサポートされていました。

The authors have nothing to disclose.

EPR分光法はネイティブに近い環境での膜タンパク質の立体構造変化を定量化するに比類のない構造的アプローチであることが証明されました。このアプローチは、私たちにX線結晶構造解析とクライオ電子顕微鏡から高解像度の構造に隠されているタンパク質のダイナミクスの分子の詳細にのぞき見することができます。しかし、他のシステムへの一般的な適用に影響を与える可能性があり?...

過去10年間、五量体リガンド依存性イオンチャネル（pLGIC）の構造的理解は、家族のいくつかのメンバーの高分解能構造の群衆に起因して、飛躍的に成長してきました。フィールド内の現在の進歩につながった主な要因は、構造決意アプローチ中の原核pLGICチャンネルの発見、真核生物の膜タンパク質の発現に1-3主要な進展、4-6と驚異的なブレークスルーを含む。7これらの構造は、上の明確なコンセンサスを提供しますpLGICの3次元構造の全体的な保全。しかし、背後に引きずるように見える二つの主要な分野は、これらのチャネル製剤の機能的特徴およびチャネル機能のメカニズムの説明があります。 ゲーティングコンフォメーション変化は複雑であり、チャネルの長さに沿って60オングストロームの距離にわたって起こり、これらの遷移は広くによって調節されます膜脂質。具体的には、負の脂質、コレステロール、およびリン脂質はpLGIC 8-11の機能を調節することが示されています。チャネル機能におけるこれらの脂質成分の正確な役割は不明のままであるが、ゲーティングの完全な分子的理解は、彼らのネイティブ環境でこれらのチャネルを研究が必要となります。部位特異的スピンラベリング（SDSL）および電子常磁性共鳴（EPR）分光法は、再構成されたシステムでは、タンパク質のダイナミクスを研究するための選択肢の技術です。 EPR分光法（蛍光分光法のように）分子の大きさ（NMRのように）、またはサンプルの光学的特性によって制限され、それによって天然の脂質状態に再構成全長構築物の測定が可能です。技術は非常に敏感であり、（ピコモルの範囲内で）比較的低いサンプル要件があります。これらの両方の側面がミリグラム以上で表現することが困難な大規模な膜タンパク質を研究するための技術は非常に適して作ります量。 部位特異的スピン標識と組み合わせて、EPR分光法を使用することは、ウェインハベルらによって開発された、およびタンパク質の種類の範囲を研究するために適合されている。12-24 EPRデータは、二次構造を調査するために使用されている、タンパク質の変化を立体構造、膜挿入深さ、およびタンパク質 - タンパク質/タンパク質 - リガンド相互作用。 この方法は、部位特異的突然変異誘発により、目的の位置にシステイン置換を含みます。部位特異的な標識化を確実にするために、システインレステンプレートを作成する別のアミノ酸（ 例えば 、セリン）で天然のシステインを置換することが必要です。これまでのところ、最も人気のあるスピン標識は、チオール特異的MTSLである：（1-オキシル2,2,5,5-テトラメチルΔ3-ピロリン-3-メチル）ジスルフィド結合のブリッジを介してタンパク質に付​​着methanethiosulfonate。その高い特異性、比較的小さいサイズ（トリプトファンよりわずかに大きい）、およびフレキシへリンカー領域の性は、このスピン標識も埋め込みシステインに優れた反応性を有することが示されています。さらに、反応性を最大化するために、タンパク質の標識反応は、界面活性剤可溶化形態で行われます。サイズ排除クロマトグラフィーにより過剰の遊離スピン標識を分離した後、タンパク質は、リポソームまたは定義された脂質組成物の二重層模倣システムに再構成されています。一般的には、システイン突然変異誘発はよくタンパク質のほとんどの地域では許容され、かつスピンプローブの比較的小さいサイズは、二次および三次構造に最小限の摂動を引き起こします。修飾は、野生型の機能を保持することを保証するために、標識され、再構成されたチャネルは、パッチクランプ測定によって研究することができます。 標識機能性タンパク質は、その後、本質的に情報の主に3つのタイプを提供する分光測定に供される：lineshにより12,14,15,20,22,23,25-27スピンプローブダイナミクスを猿の分析;常磁性緩和剤に対するプローブのアクセス;距離分布27 EPR距離は、2つの異なるアプローチによって測定されます。最初は（8 - 20Åの距離範囲）をスピン標識との間の双極子相互作用に起因するスペクトルの広がりは連続波（CW）技術に基づいている。距離を決定するために使用されている28,29秒パルス、EPRであります距離測定は、二重電子電子共鳴（DEER）で70Å。30-34にまで拡張することができる方法は、スピンエコー振幅の振動は、距離と距離の分布を決定するために分析されます。ここでのスピンエコーは双極子相互作用の周波数で変調されます。一緒に、これらのパラメータは、タンパク質のトポロジーは、二次構造要素、およびタンパク質コンホメーション変化を決定するために使用されます。

<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.

スピン標識GLICの変異体の生化学的特徴 培養の12ミリグラム/ L - 上述のプロトコールに従って、典型的には10の範囲でGLIC-MBP融合タンパク質を生じます。この値は、特にタンパク質内に埋め込ま位置について、異なる変異体を横切って変化することができるが、収率が著しく損なわれる可能性があります。これ...

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.

サイト監督スピン標識および五量体リガンド依存性イオンチャネルのEPR分光研究

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 ビュー.  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.

ビデオ: 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.

生体分子構造決定のための単分子レベルでの高精度FRET

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

生化学

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.

精製と TRPV1 の分光学的解析のための再構成

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.

核磁気共鳴分光法 (NMR) とマイクロ働く (MST) による球状と糸状のタンパク質の相互作用を測定

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.

発光共鳴エネルギーは、哺乳動物細胞で発現膜タンパク質の立体構造変化を研究するために転送

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

生物工学

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.

インタクトな機能的な膜タンパク質へのヌクレオチド結合をリアルタイムで測定

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.

天然的に無秩序なタンパク質の自己会合の検出と特性評価のための常磁性緩和増強

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>固体NMRによる天然膜タンパク質の高分解能研究

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.

固体NMRによる天然膜タンパク質の高分解能研究

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.

1分子FRETを用いた膜受容体の立体構造ダイナミクスの可視化

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.

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

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

サイト監督スピン標識および五量体リガンド依存性イオンチャネルのEPR分光研究

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固体NMRによる天然膜タンパク質の高分解能研究

固体NMRによる天然膜タンパク質の高分解能研究

1分子FRETを用いた膜受容体の立体構造ダイナミクスの可視化

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ