この作品は、全米科学財団（MCB 1021831、JM）とJ.＆E.生物医学研究プログラム（保健フロリダ部、JM）によってサポートされていました。

1。試料調製<ol><li>サンプル調製、不要なアンケージングを防ぐために、暗室内のすべてのサンプル操作を行ってください。 </li><li> 50 mMのHEPESでN、N、N &#39;、N&#39; -テトラキス[（オキシカルボニル）メチル] -1,2 -エタンジアミン） - （2 -ニトロ-4,5 -ジメトキシフェニル） - DM-nitrophen（（1を可溶化バッファー、100mMのKCl、400μMの最終濃度pHは7.0（ε350nmで = 4330 M -1 cm -1の9）。 </li><li> [DM-nitrophen]：の[Ca 2 +]の望ましい比率を達成するために、0.1M原液からのCaCl 2を追加します。 10μMを超えるCa 2 +の関連についてのK dでタンパク質については、の[Ca 2 +]の比1:1の[DM-nitrophen]はアンケージドDM-nitrophenに写真リリースのCa 2 +の結合を防ぐことが望ましい。実際に、カルシウムの90％がタンパク質に結合〜10 nMおよび400 mMのようにするDM-nitrophenおよびCa 2 +の合計濃度であることがDM-nitrophenためのK d値を考慮したK dで=10μMのはapoformになります。一方、K dは 、&lt;10μMのタンパク質への結合のCa 2 +の研究のためには、の[Ca 2 +]低下することが好ましいのとおりでのCa 2 +の錯体形成を防止するために0.95 [DM-nitrophen]比はアポタンパク質ケージ光解離前。 </li><li>参照化合物、K 3 [鉄（III）（CN）6]試料と同じ緩衝液中のNa又は2のCrO 4を可溶化する。 </li></ol> 2。実験のセットアップ<ol><li>基本的な実験構成を図2に示す。 </li><li> 1mmのプローブビームの直径（He-Neレーザーの632nmの出力を、〜5mWのレーザパワー）を調整し、温度が配置セルの中心を通ってプローブ光を伝播させるピンホール（P 2）を使用M 1ミラーを使用して制御セルホルダー。 </li><li>プローブビームを中央にサンプルの後ろにミラー（M 2）を使用します位置敏感型検出器の中心に。 </li><li>検出器の左右側の上部の2つのダイオードと底部つのダイオード間の電圧差ならびに2つのダイオード間の電圧の差があるように検出器の中心にプローブビームを集束ゼロ。 </li><li> 2 355nmのレーザミラーの間に配置され、3mmのピンホール（P 1）を用いてYAGレーザ、FWHM 5ナノ秒）：続いて、ポンプ光、Q-スイッチNdの355nmの出力の直径を形作る。 </li><li> 図2に示されているように、キュベットの中心を通ってポンプビームをCopropagate。これは、両方のレーザビームが測定可能な偏向角を得るために、ほぼ同一直線上に同様に光学セルの中心を通って伝播しPBD信号の振幅、高されていることが重要である。実験条件下で、プローブとポンプビームの交差角が15°未満である。 </li><li>プローブおよびポンプを整列させる参照化合物を使用するより長い時間スケール（〜100ミリ秒）で良好なS / N比や安定したPBD振幅、すなわち 、良好なPBD信号を達成するためにビーム。 </li><li> 355nmのレーザミラーの増分調整することによって、プローブビームに対してポンプビームの位置を調整する。 </li><li>位置検出器上の2つの頂部と底部のフォトダイオードの間の差としてPBD基準信号の振幅を測定する。 PBD信号は速い時間スケール（&lt;10秒）での振幅の急激な増加を示し、 図3で示したように100ミリ秒の時間スケールで安定して維持する必要があります。 PDB振幅の変動に撮影されたシュートは、信号振幅の5％以内であり、信号の再現性は、主に振動によって影響される。 </li><li>吸収された励起レーザパワーオン·アインシュタインの数のPBD信号の線形依存性を、E a を測定することにより放出される熱エネルギーに対してPBD信号振幅の線形性を確認するAは、励起波長での吸光度を基準に相当=（1-10-A）。 </li><li>レーザパワー下記約1,000μJと多光子吸収とそれぞれポンプ光パワーの低下を防止する励起波長が0.5未満での試料/基準化合物の吸光度を維持し、PBD信号の線形性を確保する。 </li></ol> 3。 PBD測定<ol><li>参考のためにPBDトレースの測定を開始します。 1.0センチメートル×1.0センチ1.0センチメートル×0.5センチメートルの石英セルに参照化合物の溶液を配置し、温度制御されたホルダーにセルを配置する。両方の経路長は、同等のPBD振幅を提供する。 </li><li> 3℃の温度増分で16〜35°Cの温度範囲における温度の関数として基準PBD信号を検出</li><li>各々の温度変化の際に、位置検出器上のプローブビームの位置を確認し、再必要に応じて、検出器の中心に位置を調整します。 </li><li> 数2に従って、[（DN / DT）/ C Pは ρ用語の関数としてPBD信号の線形性を確認してください。 </li><li>基準測定のための光学セルの同じ向きを維持参照化合物と同様の光学セル内の試料溶液を置く。 </li><li> PBDは、基準と同じ温度範囲でトレースし、[（のdn / dt）は/ C pが ρ用語に対して試料PBD振幅の線形性を確認する試料を検出する。 </li></ol> 4。データ解析偏向の大きさは、 式1に従って試料加熱（ΔVth）を非熱的及び体積変化（ΔVのnonth）による体積変化に正比例する。 <img src="/files/ftp_upload/50969/50969eq1.jpg" alt="式（1）" /> サンプルの振幅（S）および参照（r）の PBD信号は、それぞれ式2及び図3を用いて説明することができる。 <img src="/files/ftp_upload/50969/50969eq2.jpg" alt="式2" /> <img src="/files/ftp_upload/50969/50969eq3.jpg" alt="式3" /> PBD信号は、計器応答パラメータ（K）に正比例し、アインシュタインの数は、（E a）に吸収される。 式（2）の最初の項は、（DN / DT）（1/ρCp）は 、Qによる溶剤中に放出された熱への信号変化に対応する。のdn / dtの項は、屈折率の温度依存性の変化を表し、ρは溶媒の密度であり、C pは熱容量である。すべてのパラメータは、蒸留水で知られており、蒸留水および適切な緩衝液中の参照化合物についてPBD信号を比較することにより、緩衝液について決定することができる。 Qは熱の量であるreturne溶媒D。 ρ（DN / Dρ）項は、10〜40℃の10の温度範囲で温度に依存しない単位、ほぼ一定である。 Δnは腹筋用語は、溶液中の吸収種の存在のために屈折率の変化に対応し、プローブビームの波長は、溶液中の任意の種の吸収スペクトルに対してずれている場合、それは無視できる程度だ。参照化合物（r）とから生じる信号は、E さhνは光子励起波長におけるエネルギー、E hはν= 355 nmの励起のための80.5キロカロリー/モルである式（3）で表される。 <ol><li> 図3に示されているようにプリトリガ及びポストトリガーPBD信号との間の差としてPBD基準信号の振幅を取る。同様に、高速（A sの 速い ）の振幅を決定し、遅い相（A sの スロー）サンプルPBD信号の。 </li><li>計器応答パラメータを排除するために、Kは、参考のためPBD信号の振幅によって試料PBD信号の振幅をスケーリングする。基準信号サンプル信号の比が式（4）を与え、のように書くことができる。 <img src="/files/ftp_upload/50969/50969eq4.jpg" alt="式4" /> </li><li>この式を使用して、溶液（Q）に放出された熱および[（S / R）Eのプロットのそれぞれ傾きと切片からの光開始反応に伴う非熱体積変化（ΔVのnonth）を決定温度依存的な[（DN / DT）（1/ρCp）]に対するHのν用語。 </li><li>高速と低速のプロセスのための反応体積とエンタルピー変化を決定するために、 式5-7に応じて適切な量子収率が観察され、ボリュームおよびエンタルピー変化をスケール。 <img sRC = &quot;/ files/ftp_upload/50969/50969eq5.jpg&quot; ALT = &quot;式5&quot; /&gt; <img src="/files/ftp_upload/50969/50969eq6.jpg" alt="式6" /> <img src="/files/ftp_upload/50969/50969eq7.jpg" alt="式7" /> 200ミリ秒〜10秒の間の時間スケールで発生する反応速度を有する多段階プロセスについては、反応の個々のステップに関連するボリュームとエンタルピー変化を決定することができる。個々のステップのための振幅および寿命が体積とエンタルピー変化の時間プロファイルを記述する関数F（t）にデータを当てはめることによって分析される。 <img alt="式8" src="/files/ftp_upload/50969/50969eq8.jpg" /> α0は、A、S、 高速に対応しており、 私は、A、S、 低速 、個々のプロセスのために対応してα、τ はどこで個々の反応工程の寿命である。 foの速度定数の温度依存性からrは、個々のプロセスは、活性化エンタルピー及びエントロピーパラメータは容易にアイリングプロットを用いて決定することができる（i = 1 から/τiの k個）。 </li></ol>

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光熱方法の背後にある物理的な原理は、光励起分子は周囲の溶剤1,12の熱加熱で、その結果、基底状態に振動緩和を経由して余分なエネルギーを消費するということです。水などの溶媒の場合、これは急激な体積膨張（ΔVth）を生成する。励起状態の分子はまた、結合開裂/形成および/ ​​または分子の（ファンデ、すなわち変化の分子の大きさを変えることができる分...

このような光音響熱量測定、光熱ビーム偏向（PDB）、およびナノ秒レーザー励起で結合された過渡回折格子などの光熱方法は、短寿命中間体1,2の時間分解研究のための過渡光分光法への強力な代替物である。そのような過渡吸収及びIR分光法などの光学技術とは対照的に、それは周囲の発色団の吸収変化の時間プロファイルを監視、光熱技術は、熱/体積変化の時間依存性を検出し、したがって、光学の時間プロファイルを調べるための貴重なツールである「サイレント」のプロセス。これまでのところ、光音響熱量測定および過渡回折格子が正常グロビン3,4、酸素センサータンパク質とリガンドの相互作用における二原子リガンドの移行を含む光誘起プロセスのコンホメーションのダイナミクスを研究するために適用されているFixL 5、6オキシダーゼ電子とヘム-銅におけるプロトン輸送AND光化学系IIだけでなく、ロドプシン7とクリプトクロム8の立体構造力学の光異性化。 内部発色団および/またはフルオロフォアを欠いている生物学的システムへの光熱技術の適用を拡大するため、PBD技術は依存して、光トリガにケージド化合物を用い、数マイクロ秒以内に、リガンド/基質濃度の増加またはより速いと組み合わせたケージド化合物に関する。このアプローチは、ダイナミクスと内部フルオロフォアまたは発色団を欠いていると商業ストップフロー機器からはアクセスできませんタイムスケール上にあるタンパク質に結合するリガンド/基質に関連する構造変化のエネルギー論のモニタリングを可能にする。ここではダウンニューロンのカルシウムセンサーのC末端ドメインにケージ化合物の熱力学を監視するためのPBDのアプリケーション、Ca 2 +のDM-nitrophen、光切断だけでなく、Ca 2 +の関連についての動態ストリーム調節エレメント拮抗モジュレータ（DREAM）が提示されている。 Ca 2 +を、光にリリース〜300秒の時定数でunphotolysedケージにCa 2 +を 10秒と再バインド内のDM-nitrophenからのものである。一方、apoDREAMの存在下で、ミリ秒の時間スケールで発生する追加の運動を観察し、タンパク質へのリガンド結合を反映している。生物系の高次構造遷移をプローブするPBDの適用は、何らかの形で楽器の難しさのために制限されている、強力かつ再現性のPBD信号を達成するためのプローブとポンプ光の例えば骨の折れる整列。しかし、計測機器のセットアップの綿密な設計、温度の正確な制御、およびプローブとポンプ光の慎重な位置合わせは、広範な上、時間分解体積とエンタルピー変化のモニタリングを可能にする一貫性と堅牢PBD信号を提供10秒で約200ミリ秒の時間スケール。また、modificなど 、同一温度、緩衝液組成、光学セルの向き、レーザパワー、下のサンプルとリファレンストレースの検出を確実にする実験手順の管理ポイントを大幅に測定された反応容量およびエンタルピーでの実験誤差を低減します。

<table><tbody><tr><td>1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-&lt;em&gt;N,N,N&amp;#039;,N&amp;#039;&lt;/em&gt;-Tetraacetic Acid</td><td>Life Technologies</td><td>D-6814</td><td>DM-nitrophen, cage calcium compound, keep stock solutions in dark to prevent photodissociation</td></tr><tr><td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, &lt;em&gt;N&lt;/em&gt;-(2-Hydroxyethyl)piperazine-&lt;em&gt;N&amp;prime;&lt;/em&gt;-(2-ethanesulfonic acid)</td><td>Sigma Adrich</td><td>0909C</td><td>HEPES buffer</td></tr><tr><td>Potassium ferricyanide (III)</td><td>Sigma Aldrich</td><td>702587</td><td>reference compound for PBD measurements</td></tr><tr><td>Sodium chromate</td><td>Sigma Aldrich</td><td>307831</td><td>reference compound for PBD measurements</td></tr><tr><td>He-Ne Laser Diode 5 mW 635 nm</td><td>Edmund Optics</td><td>54-179</td><td>use as a probe beam for PBD measurements</td></tr><tr><td>Oscilloscope,&amp;nbsp;</td><td>LeCroy</td><td>Wave Surfer 42Xs</td><td>400 MHz bandwith</td></tr><tr><td>Nd:YAG laser</td><td>Continuum</td><td>ML II</td><td>pump beam for PBD measurements</td></tr><tr><td>M&lt;sub&gt;355&lt;/sub&gt;; Nd:YAG laser mirror</td><td>Edmund Optics</td><td>47-324</td><td>laser mirror for 355 nm laser line</td></tr><tr><td>M&lt;sub&gt;1&lt;/sub&gt; and M&lt;sub&gt;2&lt;/sub&gt;; Laser diode mirror</td><td>Edmund Optics</td><td>43-532</td><td>visilbe laser flat mirror, wavelength range 300-700 nm</td></tr><tr><td>P&lt;sub&gt;1&lt;/sub&gt; and P&lt;sub&gt;2&lt;/sub&gt;; Iris Diaphragm</td><td>Edmund Optics</td><td>62-649</td><td>pin hole to shape the probe and pum beams</td></tr><tr><td>L&lt;sub&gt;1&lt;/sub&gt;; bi-convex lens</td><td>Thorlabs</td><td>LB1844</td><td>a lens to focus the probe beam at the detector, EFL 50 mm, wavelength range 350-2,000 nm</td></tr><tr><td>DM, dichroic mirror</td><td>Thorlabs</td><td>DMLP505</td><td>a longpass dichroic mirror with a cutoff wavelength of 505 nm</td></tr><tr><td>F&lt;sub&gt;1&lt;/sub&gt;; Edge filter</td><td>Andower</td><td>500FH90-25</td><td>a long pass filter with a cutoff wavelength of 500 nm</td></tr><tr><td>Temperature-controlled cuvette holder</td><td>Quantum Northwest</td><td>FLASH 300</td><td>&amp;nbsp;</td></tr></tbody></table>

submillisecond conformational changes in proteins resolved by photothermal beam deflection

一緒に光音響熱量測定および熱格子と光熱ビーム偏向は、伝統的なストップを使用してアクセスできない時間スケールをミリ秒、マイクロ秒でのタンパク質の立体構造変化を誘起光の時間プロファイルボリュームとエンタルピー変化を監視する光熱方法のファミリーに属するフローの楽器。さらに、体積および/またはエンタルピーの全体的な変化がプローブされているので、これらの技術は、タンパク質およびフルオロフォアおよび発色団又はラベルを欠く他の生体高分子に適用することができる。カルシウムトランスデューサ、このような神経細胞のカルシウムセンサー、ケージカルシウム化合物、DM-nitrophenへの結合のCa 2 +に関連した構造変化のダイナミクスとエネルギー論を監視するには、フォトトリガーに遊離カルシウムの急速な（τ&lt;20秒）増加して採用されている濃度及び関連するボリュームとエンタルピー変化は、光熱ビーム偏向技術を用いてプローブする。

PBDの代表的な例は、Ca 2 + DM-nitrophenからのCa 2 +放出光は、図3に示されているトレース。遅い位相がCA 2 + nonphotolysedケージとの結合を反映しているのに対し、FASTフェーズは、Ca 2 +のDM は 、nitrophenおよびCa 2 +の解放の光切断に対応しています。温度依存係数[C のpρ/（のdn / dt）が〕の関数として基準化合物の大きさにスケーリ...

Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

光熱ビーム偏向によって解明されるタンパク質のミリ秒未満の構造変化 |化学 |ビデオ

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

ここでは、下流の調節エレメント拮抗モジュレータ、マイクロ秒およびミリ秒ダイナミクスおよびニューロンカルシウムセンサーへのカルシウムアソシエーションに関連付けられている構造変化のエネルギー論をモニターするために、DM-nitrophen、ケージドカルシウム化合物と組み合わせて光熱ビーム偏向技術の適用を報告。

光熱ビーム偏向によって分離したタンパク質におけるミリ秒以下構造変化

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

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8.9K ビュー.  Florida International University. ここでは、下流の調節エレメント拮抗モジュレータ、マイクロ秒およびミリ秒ダイナミクスおよびニューロンカルシウムセンサーへのカルシウムアソシエーションに関連付けられている構造変化のエネルギー論をモニターするために、DM-nitrophen、ケージドカルシウム化合物と組み合わせて光熱ビーム偏向技術の適用を報告。 

ビデオ: Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.

We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

分子適合体とクラスターの空間的分離

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

工学

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

感光性タンパク質中のプロトン移動とプロテイン立体配座ダイナミクスの時間分解ステップスキャンフーリエ変換赤外分光法により

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

Direct Imaging of Laser-driven Ultrafast Molecular Rotation

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb explosion imaging setup in which a hitherto-impractical camera angle is realized. In our imaging technique, diatomic molecules are irradiated with a circularly polarized strong laser pulse. The ejected atomic ions are accelerated perpendicularly to the laser propagation. The ions lying in the laser polarization plane are selected through the use of a mechanical slit and imaged with a high-throughput, 2-dimensional detector installed parallel to the polarization plane. Because a circularly polarized (isotropic) Coulomb exploding pulse is used, the observed angular distribution of the ejected ions directly corresponds to the squared rotational wave function at the time of the pulse irradiation. To create a real-time movie of molecular rotation, the present imaging technique is combined with a femtosecond pump-probe optical setup in which the pump pulses create unidirectionally rotating molecular ensembles. Due to the high image throughput of our detection system, the pump-probe experimental condition can be easily optimized by monitoring a real-time snapshot. As a result, the quality of the observed movie is sufficiently high for visualizing the detailed wave nature of motion. We also note that the present technique can be implemented in existing standard ion imaging setups, offering a new camera angle or viewpoint for the molecular systems without the need for extensive modification.

We present a protocol for creating a real-time movie of a molecular rotational wave packet using a high-resolution Coulomb explosion imaging setup.

レーザー駆動の超高速分子回転のダイレクトイメージング

We present a method for visualizing laser-induced, ultrafast molecular rotational wave packet dynamics. We have developed a new 2-dimensional Coulomb ...

化学

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

光応答性液晶の構造ダイナミクスを観察する手法

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

微結晶の時間分解フェムト秒シリアル結晶学 x 線レーザーのための高粘度押出を改善

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...

Fixed Target Serial Data Collection at Diamond Light Source

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.

We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.

ダイヤモンド光源での固定ターゲットシリアルデータ収集

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is ...

生化学

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

時間分解タンパク質誘導蛍光強化を利用して、一度に1つのαシヌクレインモノマーの安定した局所構造を同定する

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

タンパク質と水和水のピコ秒ナノ秒ダイナミクスを研究するための高分解能中性子分光法

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes can alter protein biophysics and reduce the usefulness of the data acquired. Plasmonic nanotweezers is a technique that uses localized surface plasmon resonance (LSPR) on gold nanostructures to enhance the electric field within a confined hotspot region. This field enhancement allows for the use of low laser powers to trap single nanoparticles far smaller than conventional optical tweezers, down to only a few nanometers in diameter, such as single proteins. Trapping of single protein molecules within the hotspot region induces a shift in the local refractive index (nprotein &#62; nwater), altering light scattering as a product of the molecule&#39;s polarisability, which is affected by its volume, shape anisotropy, and refractive index. An avalanche photodiode (APD) collects the subsequent changes in light scattering. These alterations can then be analyzed to determine changes in the trapped molecule, including its size, global conformation, and dynamics of conformational change over time. The incorporation of microfluidics within the system allows for controlled environmental changes and real-time monitoring of their subsequent effects on the molecule. In this protocol, we demonstrate the steps to trap single protein molecules, alter their environmental solution conditions, and monitor their corresponding conformational changes using a plasmonic nanotweezers system.

Plasmonic nanotweezers use localized surface plasmon resonance in gold nanostructures to trap single nanoparticles, including proteins, within a nanometer-scale optical field. Changes in the scattered signal reveal protein presence and conformational dynamics, enabling monitoring without fluorophore modifications or surface tethering.

プラズモニックナノピンセットを用いた単一非修飾タンパク質のコンフォメーションダイナミクスのモニタリング

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

蛍光顕微鏡によるせん断流下の単分子の立体構造変化の特徴

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...