This work has been funded by the National Research Foundation grant NRF-CRP4-2008-02 (J.T.) and Tier 1 grant RG 51/13.

<ol>
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The authors have nothing to disclose.

本論文では、平衡沈降を使用して、洗剤の小さな膜タンパク質のオリゴマー化の試料調製および分析のための実験プロトコルを提供します。密度マッチングステップが必要とされないように記載されるプロトコルは、可溶性タンパク質に同等に有効で-and simpler-ある。実際に、システムは、界面活性剤とタンパク質との混合物で構成されている。それは、粒子浮選に寄与しないように、沈降研...

分析超遠心分離1-5は弱いと強い相互作用の両方にアクセス可能であること、生理的条件下での高分子の相互作用を研究するための最も重要な方法の一つである。この方法は、無標識であり、光吸収または干渉を利用し、さらに蛍光光学システムは、大きさが6の数桁にわたって濃度範囲にアクセスするために使用することができる。 ほとんどの生化学的過程が可逆的相互作用に依存するため、この方法は特に便利です。これらの相互作用の化学量論および強度を定量的生物学的プロセスを理解することを特徴とする必要があり、多数の方法が、この目的の7,8のために存在する。しかし、一時的な相互作用は、9を研究することは困難である。 巨大分子の相互作用を特徴付けるための方法の選択は、その静的または動的な性質に依存する。最初のケースでは、sedim<html> entation速度（SV）は、半径方向の輸送速度が測定され、複合体が浮力質量と形状の違いに基づいて分画される場合、使用されている。 対照的に、実験の時間スケールで可逆的で動的な関連付けは、物理的に分離することができない。この場合には、自己または非共有結合性相互作用をもたらすヘテロ相互作用は、総タンパク質濃度に依存して平衡状態にある。これらの動的な相互作用は、沈降平衡（SE）及び沈降速度（SV）10の両方で研究することができる。しかし、第一の方法は、実行するために簡単であり、ここで説明されている。平衡拡散沈降の間に到達するようにSEでは、遠心分離が十分に低速で行われる。この時点で、半径方向距離の関数としての光信号（UV-VIS）の平衡プロファイルは、アソシエーション11用に事前設定された熱力学モデルを用いて分析することができる。 ve_content &quot;&gt;本研究では、沈降平衡試験があるため、その疎水性のイオンチャネルを形成し、ウイルス膜タンパク質の自己会合が示されているが、実験は、界面活性剤の存在下で実行され、この場合の密度のさ溶媒は、界面活性剤のそれに一致させる必要があるが、記載されるプロトコルは、水溶性タンパク質の場合には同一の場合と、無溶媒濃度マッチングを必要としないであろうことを除いて。 使用されるタンパク質は、世界中のヒト呼吸器合胞体ウイルス（はhRSV）、乳児の下気道疾患の原因となるパラミクソウイルス科に包まニューモ、高齢者や免疫不全の集団12でエンコードされます。最大6400万はhRSV感染16万人の死亡の症例が毎年発生すると報告した。 はhRSVゲノム三膜タンパク質F、G、及び小さな疎水性（SH）を含む11のタンパク質を転写する。 SHタンパク質が関与しているRSV感染の病因に。 SH遺伝子（RSVΔSH）を欠くRSVは、生存可能であったシンシチウムの形成を引き起こし、野生型（WT）ウイルス13-16と同様に増殖した。しかし、RSVΔSHウイルスは上気道15、16にWTよりも低い効率で10倍に複製。また、RSVΔSHウイルスはin vivoでのマウスとチンパンジーモデル13、17で減衰されました。 SHタンパク質は、主にゴルジコン ​​パートメント18の膜64に蓄積する（RSVサブグループA）または65（RSVサブグループB）アミノ酸長いII型内在性膜タンパク質である。 SHタンパク質は高度に保存されている20,21を単一の予測ヘリックス膜貫通（TM）ドメイン19を有している。 C-およびN-末端膜外ドメインは、それぞれ、内腔に/細胞外及び細胞質に配向されている。 両方の合成TMドメイン（残基18-43）及び全長SHタンパク質は、界面活性剤の様々なhomopentamersを形成することが示されている。ホモ五量体の形は、平面脂質二重層22,23におけるチャネル活性を担当しています。脂質二重層におけるTMモノマーの正しい配向は、第1のインターらせん配向に近い内腔であることのHis-22を示した部位特異的な赤外線二色23を使用して決定した。同一のTMドメイン配向は、ドデシルホスホコリンにおける全長タンパク質の五量ヘリックスバンドル（DPC）が22をミセルが再構成されたNMR研究により確認された。この「ミセル」モデルでは、単一のA-らせんTMドメインは、拡張B-ヘアピンによってC末端AヘリックスによってN末端が隣接し、そしてた。 SHタンパク質の2つのプロトン化可能な残基のHis-22およびHis-51、TMドメイン（内腔に配向）に配置され、膜外C末端βヘアピンの先端のそれぞれ、（遠チャンネル孔から）。バイセルエンバイロでnmentは、しかし、TMのαヘリックスは、彼の-51にまで延びており、両方のHis残基は、チャンネル24の内腔にアクセス可能である。チャネル構造は、（のCys-45へのSer-29）狭い地域漏斗のようなアーキテクチャ22は、22は疎水性側鎖（イル·32、イル= 36、イル= 40およびLeu-44）が並んで採用しており、イル·36は、チャンネル内腔に狭いポイントを定義します。のHis-51が最も小さい開口部の先端部に、一方のHis-22は、このファンネルの最大の開口部に配置されている。 本論文では、沈降平衡モードでの分析の遠心分離は、Hisプロトン化は、SHタンパク質五量の安定性に影響を与えるかどうかを決定するために使用されてきた。この場合、SHタンパク質は、そのSHタンパク質の形態五量体オリゴマー22を表示するために以前に使用されているC14-ベタイン界面活性剤で可溶化した。

<table><tbody><tr><td>3-(N,N-dimethylmyristylammonio)propanesulfonate</td><td>Sigma</td><td>T0807</td><td></td></tr><tr><td>Deuterium oxide 99.8%</td><td>Cambridge Isotope</td><td>DLM-4-99.8</td><td></td></tr><tr><td>An-50 Ti Rotor, Analytical, 8-Place</td><td>Beckman Coulter</td><td>363782</td><td></td></tr><tr><td>An-60 Ti Rotor, Analytical, 4-Place</td><td>Beckman Coulter</td><td>361964</td><td></td></tr><tr><td>Cell housing</td><td>Beckman Coulter</td><td>334784</td><td></td></tr><tr><td>12 mm six-channel centerpiece, epon charcoal-filled</td><td>Beckman Coulter</td><td>331376</td><td></td></tr><tr><td>Window holder</td><td>Beckman Coulter</td><td>305037</td><td></td></tr><tr><td>Window gasket</td><td>Beckman Coulter</td><td>327021</td><td></td></tr><tr><td>Window liner</td><td>Beckman Coulter</td><td>362329</td><td></td></tr><tr><td>Sapphire window</td><td>Beckman Coulter</td><td>307177</td><td></td></tr><tr><td>Quartz window</td><td>Beckman Coulter</td><td>301730</td><td></td></tr><tr><td>Screw-ring washer</td><td>Beckman Coulter</td><td>362328</td><td></td></tr><tr><td>Screw ring</td><td>Beckman Coulter</td><td>301922</td><td></td></tr><tr><td>Spinkote</td><td>Beckman Coulter</td><td>306812</td><td></td></tr><tr><td>Torque stand assembly</td><td>Beckman Coulter</td><td>361318</td><td></td></tr><tr><td>Counterbalance</td><td>Beckman Coulter</td><td>360219</td><td></td></tr><tr><td>Cell alignment tool</td><td>Beckman Coulter</td><td>362340</td><td></td></tr><tr><td>SEDNTERP</td><td></td><td></td><td>http://bitcwiki.sr.unh.edu/index.php/Main_Page</td></tr><tr><td>HeteroAnalysis&amp;nbsp;</td><td></td><td></td><td>http://www.biotech.uconn.edu/auf/?i=aufftp</td></tr><tr><td>SEDFIT</td><td></td><td></td><td>http://www.analyticalultracentrifugation&lt;br/&gt; .com/sedfit.htm</td></tr><tr><td>SEDPHAT</td><td></td><td></td><td>http://www.analyticalultracentrifugation&lt;br/&gt; .com/sedphat/default.htm</td></tr></tbody></table>

sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability

分析用超遠心分離（AUC）は、相互作用の強さの広い範囲にわたって、生理的条件下において高分子間の可逆的な相互作用を研究するために使用することができる。これはAUC定量的に化学量論および生化学過程で一過性で可逆的でホモおよびヘテロ会合の熱力学を評価するための最適な方法になります。沈降平衡（SE）のモダリティでは、拡散と沈降の間のバランスは、特定の相関モデルに依存して半径方向距離の関数としてプロファイルを提供しています。本明細書において、詳細なSEプロトコルは、分析用超遠心を用いた小型膜タンパク質のオリゴマーのサイズ及びモノマー - モノマー関連エネルギーを決定するために記載されている。 AUC-ESは、物理的原理に基づいて、無標識であり、そして水溶性及び膜タンパク質の両方で使用することができる。例は後者の表示され、人間の呼吸器合胞体ウイルスの小さな疎水性（SH）タンパク質（はhRSV五量体イオンチャネルを形成する単一のαヘリックス膜貫通（TM）ドメインを持つ）、65アミノ酸のポリペプチド。 NMRベースの構造データは、SHタンパク質がチャネルの内腔に面して配向され、その膜貫通ドメイン二つのプロトン化のHis残基を有することを示している。 SE実験は、pHが会合定数及びSHタンパク質のオリゴマーサイズにどのように影響するかを決定するために設計されています。五量体形態はすべての場合に保存されたが、その結合定数が低いpHで減少した。これらのデータは、SHタンパク質内の2つのHis残基の内腔の向きと一致SHチャネル活性について観察同様のpH依存性と一致している。後者は、静電反発力が発生し、低pHでのオリゴマーの安定性を低減することができる。要約すると、この方法が適用されるたびに、生理学的条件の微妙なタンパク質 - タンパク質会合の変化に関する定量的情報を測定する必要がある。

50mMのトリスC14SBの界面活性剤ミセルの径方向分布プロフィール、100mMのNaCl pH7.3の形の線形モデル（ 図7A）に装着することができ、非常に浅い指数関数。この分布の傾きは逆にD 2 O濃度（ 図7B）と相関している。傾きがゼロとなる点、 すなわち、マッチングD 2 O濃度は、32.3％であることが見出された。 以下の図7を</str...

Watch this Scientific Journal Video about Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability at JoVE.com

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

小オリゴマー形成膜タンパク質の沈降平衡：五量体安定性に対するヒスチジンプロトン化の影響

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

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10.5K ビュー.  Nanyang Technological University. Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

ビデオ: Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

水素交換質量分析法を用いてタンパク質のダイナミクスを分析する

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

化学

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

蛍光分光法により測定膜タンパク質フォールディングの熱力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

生物工学

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

透過型電子顕微鏡により膜相互作用タンパク質を視覚化し、分析するための方法

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

生化学

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Channels for the transportation of water molecules in enzymes influence active site solvation and catalysis. Herein we present a protocol for the engineering of these additional catalytic motifs based on in silico computer modeling and experiments. This will enhance our understanding of the influence of solvent dynamics on enzyme catalysis.

膜酵素で溶剤ダイナミクスによって誘導される解明エントロピーレートの高速化

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

3色 1 分子 FRET を使用して蛋白質の相互作用の相関関係を研究するには

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

洗剤無料補完充電プロテオリポソーム融合ペプチドを用いた脂質膜に膜タンパク質の超高速再構成。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

膜蛋白質の構造解析および経験的モデリング中性子小角散乱実験でコントラストに一致する洗剤

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X線結晶学は 、熱利子マリティマ M42アミノペプチダーゼTmPepT050のオリゴマー状態推移を研究する

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

遺伝学

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.

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.

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

小オリゴマー形成膜タンパク質の沈降平衡：五量体安定性に対するヒスチジンプロトン化の影響

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水素交換質量分析法を用いてタンパク質のダイナミクスを分析する

蛍光分光法により測定膜タンパク質フォールディングの熱力学

透過型電子顕微鏡により膜相互作用タンパク質を視覚化し、分析するための方法

膜酵素で溶剤ダイナミクスによって誘導される解明エントロピーレートの高速化

3色 1 分子 FRET を使用して蛋白質の相互作用の相関関係を研究するには

洗剤無料補完充電プロテオリポソーム融合ペプチドを用いた脂質膜に膜タンパク質の超高速再構成。

膜蛋白質の構造解析および経験的モデリング中性子小角散乱実験でコントラストに一致する洗剤

X線結晶学は、熱利子マリティマ M42アミノペプチダーゼTmPepT050のオリゴマー状態推移を研究する

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

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

小オリゴマー形成膜タンパク質の沈降平衡：五量体安定性に対するヒスチジンプロトン化の影響

要約

さらに動画を探す

この動画の章

水素交換質量分析法を用いてタンパク質のダイナミクスを分析する

蛍光分光法により測定膜タンパク質フォールディングの熱力学

透過型電子顕微鏡により膜相互作用タンパク質を視覚化し、分析するための方法

膜酵素で溶剤ダイナミクスによって誘導される解明エントロピーレートの高速化

3色 1 分子 FRET を使用して蛋白質の相互作用の相関関係を研究するには

洗剤無料補完充電プロテオリポソーム融合ペプチドを用いた脂質膜に膜タンパク質の超高速再構成。

膜蛋白質の構造解析および経験的モデリング中性子小角散乱実験でコントラストに一致する洗剤

X線結晶学は 、熱利子マリティマ M42アミノペプチダーゼTmPepT050のオリゴマー状態推移を研究する

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

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

X線結晶学は、熱利子マリティマ M42アミノペプチダーゼTmPepT050のオリゴマー状態推移を研究する