本研究の一部はBMWiとBMBFプロジェクト」Aptasens」（BMBF / DLR 01RB0805A）が資金を提供し、IGF-プロジェクト「S-ふるい」（490 ZBG / 1）によって資金を供給されました。英語のネイティブスピーカーとして原稿を読み取るためのAFM研究中およびエリックV.ジョンストンへの彼の貴重な助けのためのトビアス・J・ギュンターに感謝します。さらに、この論文の著者は、Manjaフォーゲル、ナンシーアンガー、カレンE. Viacavaとヘルムホルツ研究所のグループバイオテクノロジー（ICP-MS測定における支援のためのリソースエコロジー研究所から）アリーンリッターとサブリナGurlitに感謝したいと思います資源技術のためのフライベルク。

著者らは、開示することは何もありません。

この研究では、異なる分析方法の組み合わせを用いて調べたS層タンパク質の金の結合を研究しました。具体的には、金の結合は、例えば 、感覚面鉱業水またはプロセス溶液から金を回収するだけでなく、材料の建設のためだけでなく、非常に魅力的です。 Auの相互作用の研究のために金（Au（III）及びAu（0）-NPs）に懸濁し、SLP1の単層を再結晶化して、タンパク質が持っていたが単離?...

原因エレクトロニクス、触媒、バイオセンサ、または医療機器のようないくつかのアプリケーションのための金の使用の増加に、この貴金属の需要はここ数年の時間6-9かけて成長してきました。重鎖または貴金属、最も環境汚染は継続的なプロセスであるが、金だけでなく、他の多くの貴重な重金属は、鉱業活動を通じて、希薄濃度の工業排水を経て環境中に放出され、廃棄物処理7,8,10されています主に技術的な活動によって引き起こされます。これは、自然の生態系の重要な干渉につながり、潜在的に人間の健康9を脅かします。これらの負の結果を知ることは、工業廃水からの金属のリサイクルに汚染された生態系や改善点から金属を除去するための新しい技術の探索を推進しています。沈殿又はイオン交換のような十分に確立された物理化学的方法には、特に高で、それほど効果的ではありませんLYは、溶液7,8,11に希釈しました。バイオソープションは、いずれかの生きているか死んバイオマスで、廃水処理10,12のための魅力的な代替です。そのような生物学的材料の使用は、毒性化学物質の消費量を低減することができます。多くの微生物は、蓄積又は金属を固定するために記載されています。例えば、Lysinibacillusのスフェリカスの細胞が（L.スファエリカス ）JG-A12は、貴金属のための高い結合能力を示している、例えば、パラジウム（II）、白金（II）、金（III）、及びPbのような他の有毒金属（II）またはU（VI）4,13、クロム（VI）14のためのバチルス・メガテリウムの細胞、金のために白金（II）およびPd（II）15、及びクロレラ下品ためのサッカロマイセス・セレビシエの細胞（III）とU（VI）16 、17。金（III）、パラジウム（II）、およびPt（II）のような前の金属の結合はまた、 デスルホビブリオ18およびL.ためのdesulfuricansについて報告されていますスフェリカス JG-B53 19,20。それにもかかわらず、ではないアルリットルの微生物は、金属を大量に結合して、収着材料としての用途は限ら12,21です。また、容量の金属結合は、異なるパラメータ、 例えば、細胞組成物、使用されるバイオコンポーネント、または環境や実験条件（pH、イオン強度、温度など）に依存します。単離された細胞壁断片22,23の研究は、膜脂質、ペプチドグリカン、タンパク質、または他の構成要素と同様に、複雑な構築全細胞8,21のプロセスを結合金属を理解するのに役立ちます。 この研究に着目セル構成要素は、S層タンパク質です。 S層タンパク質は、多くの細菌及び古細菌の外側細胞エンベロープの一部であり、それらは、約15を構成する - これらの生物の全タンパク質質量の20％。環境への最初のインターフェイスとして、これらの細胞の化合物は、強く、細菌の吸着特性3に影響を与えます 。 40の範囲の分子量を有するS層タンパク質kDaでの数百の細胞内で生産され、それらは脂質膜または高分子の細胞壁成分に層を形成することができる場合に外部組み立てられます。一旦単離されると、ほぼ全てのS層タンパク質は自発的に界面で、または平面または管状の構造体3を形成する面に、懸濁液中で自己集合する固有の特性を有しています。タンパク質の単分子層の厚さは、細菌に依存し5の範囲内である- 25nmで24。一般に、形成されたS層タンパク質の構造は、35ナノメートル3,24に対して斜め（P1またはP2）、正方形（P4）、または六角形（P3またはP6）2.5の格子定数との対称性を有することができます。格子形成は、二価の陽イオンに依存して、主のCa 2+ 25,26、ラフ、Jらに多くのケースであるように思われます。 S層タンパク質ベースのエンジニアナノ構造における産業用アプリケーションに基づくナノコンポジット。 （編Tijana Z.グローブ＆Aitziber L. Cortajarena）（スプリンガー、2016（提出））。それにもかかわらず、特に例えば、Ca 2+およびMg 2+などの二価カチオンのモノマーの折り畳み、モノマー-モノマー相互作用、格子を形成し、異なる金属の役割の完全な反応カスケードは、まだ完全には理解されていません。 グラム陽性菌株L. 27（新系統発生的分類した後、バチルス・スファエリカスから改称） スフェリカス JG-B53はウラン鉱山廃棄物の山「ハーバーランド」（Johanngeorgenstadt、ザクセン、ドイツ）4,28,29から単離しました。その機能S層タンパク質（SLP1）は正方格子、116 kDaで30の分子量、および細菌細胞31生きに ​​≈10nmの厚さを有しています。以前の研究では、約10nmの厚さで閉じ、安定したタンパク質層のインビトロでの形成は10分未満19で達成されました。関連菌株L.スフェリカス JG-A12は、また「ハーバーランド &quot;パイルからの分離株は、高い金属結合能力を有し、その単離されたS層タンパク質は、Auのような貴金属のための高い化学的および機械的安定性と優れた吸着率を示した（III）、白金（II）、およびPd（II）4,32,33。貴金属のこの結合は、いくつかの金属について多かれ少なかれ特異的であり、ポリマーの外側及び内側タンパク質表面上及び細孔内の官能基、イオン強度、およびpH値の利用可能性に依存します。 、OH - 、PO 4 - - 、SO 4 - 、およびSO-タンパク質による金属相互作用に関連する官能基は、COOH-、NH 2です。原理的には、金属結合能力は、アプリケーション、 ラフ、Jらの広いスペクトルを開きます。 S層タンパク質ベースのエンジニアナノ構造における産業用アプリケーションに基づくナノコンポジット。 （編Tijana Z.グローブ＆Aitziber L. Cortajarena）（スプリンガー、2016年（提出））。 例えば、除去又は回復のためbiosorptiveコンポーネントとして溶解毒性または有価金属の、合成または触媒反応、およびバイオ感覚層3,5,18,33のような他の生体工学材料のため、定期的に構造化金属ナノ粒子（NPS）の定義された堆積のためのテンプレート。 Auのような規則的に配置されたNPアレイ（0）-NPsはCO酸化34-37ための分子エレクトロニクスやバイオセンサー、超高密度記憶装置、および触媒に至るまでの主要な用途のために使用することができます。このようなアプリケーションや、これらの材料のスマートなデザインの開発は、基礎となる金属結合メカニズムのより深い理解を必要とします。 このようなバイオベース材料の開発のための前提条件は、生体分子と技術面38,39との間の界面層の信頼できる実装です。例えば、高分子電解質は、S層タンパク質39の再結晶化のための界面層として使用されているレイヤー・バイ・レイヤー（のLbL）技術40,41に組み付け</SUP&gt;。このようなインタフェースは、再現可能かつ定量的な方法でタンパク質コーティングを実行するための比較的簡単な方法を提供しています。接着剤プロモーターと変更することなく、種々の実験を行うことで、コーティング速度、層の安定性、および生体分子19,42、ラフ、J.らと金属との相互作用に関する記述することが可能です。 S層タンパク質ベースのエンジニアナノ構造における産業用アプリケーションに基づくナノコンポジット。 （編Tijana Z.グローブ＆Aitziber L. Cortajarena）（スプリンガー、2016年（提出））。しかし、タンパク質吸着およびタンパク質 - 表面相互作用の複雑なメカニズムは完全に理解されていません。特に立体構造、パターンの向き、コーティング密度の情報がまだ不足しています。 消費モニタリング（QCM-D）技術で水晶マイクロバランスは、タンパク質吸着、コーティング速度を研究するためのツール、および対話プロとして近年注目を集めていますナノメートルスケール19,43-45上セス。この技術は、リアルタイムで大量の吸着の詳細な検出を可能にし、タンパク質格子19,20,42,46-48に機能性分子のタンパク質自己集合プロセスと結合するための指標として用いることができます。また、QCM-Dの測定は、天然の生物学的条件下でのタンパク質性の層と金属の相互作用の過程を研究する可能性を開きます。最近の研究において、Euのような選択された金属とS層タンパク質の相互作用（III）、金（III）、パラジウム（II）、およびPtは（II）QCM-D 19,20で検討されています。吸着したタンパク質層は、グラム陽性細菌の細胞壁の単純化モデルとしての役割を果たすことができます。この単一の成分の研究では、金属相互作用のより深い理解に寄与することができます。しかし、単にQCM-D実験は、タンパク質に金属の表面構造と影響に関する記述を許可していません。他の技術は、そのような情報を得るために必要です。 One POSイメージングバイオナノ構造のための任及び構造特性情報を取得するには、原子間力顕微鏡（AFM）です。 提示研究の目的は、特にL. SLP1に、S層タンパク質に金（Au（III）とAu（0）-NPs）の収着を調査することでしたスフェリカス JG-B53。 ICP-MSを使用して5.0とQCM-Dを使用して固定化されたS-層と - 実験は、2.0のpH範囲内のバッチスケールで懸濁タンパク質を用いて行きました。さらに、格子の安定性に対する金属塩溶液の影響は、その後のAFM研究で調査しました。これらの技術の組み合わせは、特定の金属親和性について細菌細胞全体のイベントを結合についての詳細を学ぶためのツールとして、in vitroでの金属相互作用プロセスのより良い理解に貢献しています。この知識だけでなく、環境保護のための金属の回収のために適用可能なフィルター材料の開発と再の保全のために重要です49だけでなく、様々な技術的なアプリケーションのための高度に秩序化された金属NPのアレイの開発のためのソース。

<table><tbody><tr><td>&lt;strong&gt;equiment and software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bioreactor, Steam In Place 70L Pilot System</td><td>Applikon Biotechnology, Netherlands</td><td>Z6X</td><td>Including dO&lt;sub&gt;2&lt;/sub&gt;, pH sensors of Applikon Biotechnology and BioXpert software V2</td></tr><tr><td>Noninvasive Biomass Monitor BugEye 2100</td><td>BugLab, Concord (CA), USA</td><td>Z9X</td><td>---</td></tr><tr><td>Spectrometer Ultrospec 1000</td><td>Amersham Pharmacia Biotech, Great Britain</td><td>80-2109-10</td><td>Company now GE Healthcare Life Sciences</td></tr><tr><td>MiniStar micro centrifuge</td><td>VWR, Germany</td><td>521-2844</td><td>For centrifugation of cultivation samples</td></tr><tr><td>Research system microscope BX-61</td><td>Olympus Germany LLC, Germany</td><td>037006</td><td>Microscope in combination with imaging software</td></tr><tr><td>Cell^P (version 3.1)</td><td>Olympus Soft Imaging Solutions LLC, M&amp;uuml;nster, Germany</td><td>---</td><td>together with microscope</td></tr><tr><td>Powerfuge Pilot Separation System Serie 9010-S</td><td>Carr Centritech, Florida, USA</td><td>9010PLT</td><td>For biomasse harvesting</td></tr><tr><td>T18 basic Ultra Turrax</td><td>IKA Labortechnik, Germany</td><td>431-2601</td><td>For flagella removal and sample homogenization</td></tr><tr><td>Sorvall Evolution RC Superspeed Centrifuge</td><td>Thermo Fisher Scientific, USA</td><td>728411</td><td>Used within protein isolation</td></tr><tr><td>Mobile high shear fluid processor, M-110EH-30 Pilot</td><td>Microfluidics, Massachusetts, USA</td><td>M110EH30K</td><td>Used for cell rupture</td></tr><tr><td>Alpha 1-4 LSC Freeze dryer</td><td>Martin Christ Freeze dryers LLC, Osterode, Germany</td><td>102041</td><td>---</td></tr><tr><td>UV-VIS spectrophotometry (NanoDrop 2000c)</td><td>Thermo Fisher Scientific, USA</td><td>91-ND-2000C-L</td><td>For determination of protein concentration</td></tr><tr><td>Mini-PROTEAN vertical electrophoresis chamber</td><td>Bio-Rad Laboratories GmbH, Munich, Germany</td><td>165-3322</td><td>For SDS-PAGE</td></tr><tr><td>VersaDoc Imaging System 3000</td><td>Bio-Rad Laboratories GmbH, Munich, Germany</td><td>1708030</td><td>Used for imaging of SDS-PAGE gels</td></tr><tr><td>ICP-MS Elan 9000</td><td>PerkinElmer, Waltham (MA), USA</td><td>N8120536</td><td>For determination of metal concentration</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern Instruments, Worcestershire United Kingdom</td><td>ZEN3600</td><td>For determination of nanoparticle size</td></tr><tr><td>Q-Sense E4 device&amp;nbsp;</td><td>Q-Sense AB, Gothenburg, Sweden</td><td>QS-E4</td><td>ordered via LOT quantum design (software included with E4 platform)</td></tr><tr><td>Q-Soft 401 (data recording)</td><td>Q-Sense AB, Gothenburg, Sweden</td><td></td><td></td></tr><tr><td>Q-Tools 3 (data evaluation and modelling)</td><td>Q-Sense AB, Gothenburg, Sweden</td><td></td><td></td></tr><tr><td>QCM-D flow modules QFM&amp;nbsp;401&amp;nbsp;</td><td>Q-Sense AB, Gothenburg, Sweden</td><td>QS-QFM401</td><td>ordered via LOT quantum design</td></tr><tr><td>QSX&amp;nbsp;303 SiO&lt;sub&gt;2&lt;/sub&gt; piezoelectric AT-cut quartz sensors</td><td>Q-Sense AB, Gothenburg, Sweden</td><td>QS-QSX303</td><td>ordered via LOT quantum design</td></tr><tr><td>Ozone cleaning chamber</td><td>Bioforce Nanoscience, Ames (IA), USA</td><td>QS-ESA006</td><td>ordered via LOT quantum design</td></tr><tr><td>Atomic Force Microscope MFP-3D Bio AFM</td><td>Asylum Research, Santa Barbara (CA), USA</td><td>MFP-3DBio</td><td>AFM measurements and imaging software</td></tr><tr><td>Asylum Research AFM Software AR Version 120804+1223</td><td>Asylum Research, Santa Barbara (CA), USA</td><td>---</td><td>imaging software included in Cat. No. MFP-3DBio</td></tr><tr><td>Igor Version Pro 6.3.2.3 Software</td><td>WaveMetrics, Inc., USA</td><td>---</td><td>imaging software included in Cat. No. MFP-3DBio</td></tr><tr><td>BioHeater</td><td>Asylum Research, Santa Barbara (CA), USA</td><td>Bioheater</td><td>Sample heater for AFM measurements</td></tr><tr><td>Biolever mini cantilever,&amp;nbsp; BL-AC40TS-C2</td><td>Olympus Germany LLC, Germany</td><td>&amp;nbsp;BL-AC40TS-C2</td><td>Prefered cantilever for AFM measurements</td></tr><tr><td>WSxM 5.0 Develop 6.5 (2013)</td><td>Nanotec Electronica S.L. , Spain</td><td>freeware</td><td>Software for AFM analysis</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Detergents and other equiment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>acidic acid, 100 %, p.A.</td><td>CARL ROTH GmbH+CO.KG</td><td>3738.5</td><td>Danger, flammable and corrosive liquid and vapour. Causes severe skin burns and eye damage.</td></tr><tr><td>Antifoam 204</td><td>Sigma-Aldrich Co. LLC.</td><td>A6426</td><td>For foam suppression</td></tr><tr><td>bromophenol blue, sodium salt</td><td>Sigma-Aldrich Co. LLC.</td><td>B5525</td><td>---</td></tr><tr><td>Coomassie Brilliant Blue R (C&lt;sub&gt;45&lt;/sub&gt;H&lt;sub&gt;44&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;NaO&lt;sub&gt;7&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;)</td><td>CARL ROTH GmbH+CO.KG</td><td>3862.1</td><td>---</td></tr><tr><td>Deoxyribonuclease II from porcine spleen</td><td>Sigma-Aldrich Co. LLC.</td><td>D4138</td><td>Typ IV , 2,000 - 6,000&amp;nbsp;Kunitz units/mg protein</td></tr><tr><td>Ethanol, 95%</td><td>VWR, Germany</td><td>20827.467</td><td>Danger, flammable</td></tr><tr><td>glycerine, p.A.</td><td>CARL ROTH GmbH+CO.KG</td><td>3783.1</td><td>---</td></tr><tr><td>Guanidine hydrochloride (GuHCl)</td><td>CARL ROTH GmbH+CO.KG</td><td>0037.1</td><td>---</td></tr><tr><td>Hellmanex III</td><td>Hellma GmbH &amp;amp; Co. KG</td><td>9-307-011-4-507</td><td>---</td></tr><tr><td>Hydrochloric acid (HCl) (37%)</td><td>CARL ROTH GmbH+CO.KG</td><td>4625.2</td><td>Danger; Corrosive, used for pH adjustment</td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma-Aldrich Co. LLC.</td><td>L6876&amp;nbsp;</td><td>Lyophilized powder, protein = 90 %, = 40,000 units/mg protein (Sigma)&amp;nbsp;</td></tr><tr><td>Magnetic stirrer with heating,&amp;nbsp; MR 3000K</td><td>Heidolph Instruments GmbH &amp;amp; Co.KG, Germany</td><td>504.10100.00</td><td>Standard stirrer within experiment</td></tr><tr><td>NB-Media DM180</td><td>Mast Diagnostica GmbH</td><td>121800</td><td>---</td></tr><tr><td>Nitric acid (HNO&lt;sub&gt;3&lt;/sub&gt;)</td><td>CARL ROTH GmbH+CO.KG</td><td>HN50.1</td><td>Danger; Oxidizing, Corrosing</td></tr><tr><td>PageRuler Unstained Protein Ladder</td><td>ThermoScientific-Pierce</td><td>26614</td><td>---</td></tr><tr><td>Poly(sodium 4-styrenesulfonat) (PSS)</td><td>Sigma-Aldrich Co. LLC.</td><td>243051</td><td>Average Mw ~70,000</td></tr><tr><td>Polyethylenimine (PEI), branched</td><td>Sigma-Aldrich Co. LLC.</td><td>408727</td><td>Warning; Harmful, Irritant, Dangerous for the environment; average Mw ~25,000</td></tr><tr><td>Potassium carbonate anhydrous (K&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich Co. LLC.</td><td>60108</td><td>Warning; Harmful</td></tr><tr><td>Ribonuclease A from bovine pancreas&amp;nbsp;</td><td>Sigma-Aldrich Co. LLC.</td><td>R5503</td><td>Type I-AS, 50 - 100 Kunitz units/mg protein&amp;nbsp;</td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck KGaA</td><td>106688</td><td>Danger; very toxic and Dangerous for the environment</td></tr><tr><td>Sodium chloride (NaCl)</td><td>CARL ROTH GmbH+CO.KG</td><td>3957.2</td><td>---</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich Co. LLC.</td><td>L-5750</td><td>Danger; toxic</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>CARL ROTH GmbH+CO.KG</td><td>6771.1</td><td>Danger; Corrosive, used for pH regulation within cultivation and pH adjustment</td></tr><tr><td>Spectra/Por 6, Dialysis membrane, MWCO 50,000&amp;nbsp;</td><td>CARL ROTH GmbH+CO.KG</td><td>1893.1</td><td>---</td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>CARL ROTH GmbH+CO.KG</td><td>HN52.2</td><td>Danger; Corrosive, used for pH regulation within cultivation</td></tr><tr><td>Tannic acid (C&lt;sub&gt;76&lt;/sub&gt;H&lt;sub&gt;52&lt;/sub&gt;O&lt;sub&gt;46&lt;/sub&gt;)</td><td>Sigma-Aldrich Co. LLC.</td><td>16201</td><td>---</td></tr><tr><td>TRIS HCl (C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;HCl)</td><td>CARL ROTH GmbH+CO.KG</td><td>9090.2</td><td>---</td></tr><tr><td>Triton X-100</td><td>CARL ROTH GmbH+CO.KG</td><td>3051.3</td><td>Warning; Harmful, Dangerous for the environment</td></tr><tr><td>VIVASPIN 500, 50,000 MWCO Ultrafiltration tubes</td><td>Sartorius AG</td><td>VS0132</td><td>---</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich Co. LLC.</td><td>M6250</td><td>Danger, toxic</td></tr></tbody></table>

au-interaction of slp1 polymers and monolayer from lysinibacillus sphaericus jg-b53 - qcm-d, icp-ms and afm as tools for biomolecule-metal studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

微生物およびSLP1キャラの育成 細菌増殖の記録されたデータは、約5時間で指数増殖期の終わりを示します。以前の研究は、それにもかかわらず、定義されたメディアコンポーネントを使用して、培養の最適化またはfed-。最大収率19 4.36グラム/ L（SLP1は収穫のこの時点から単離することができることを?...

Watch this Scientific Journal Video about Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies at JoVE.com

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

SLP1ポリマーと単層のAuのインタラクションから<em&gt; Lysinibacillusスフェリカス</em&gt; JG-B53 - 生体分子金属研究のためのツールと​​して、QCM-D、ICP-MSおよびAFM

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

au-interaction-slp1-polymers-monolayer-from-lysinibacillus-sphaericus

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

11.3K ビュー.  Helmholtz-Zentrum Dresden-Rossendorf. To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

ビデオ: Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

その場 SIMSおよび質量選択されたイオンのソフトランディングで調製し明確に定義された表面の赤外分光法における

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

化学

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

原子間力分光法による単一分子の接着性を調査

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

工学

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

動的タンパク質複合体の分析の組み立てし、質量分析法と電子顕微鏡を用いた Biolayer 干渉法によるバイオ センサーから解放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

生化学

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

単一分子力分光法を用いて無機材料とアミノ酸とペプチドの相互作用への洞察

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1-媒介酵素合成と単分子力分光法のための重合タンパク質の固定化

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子間力顕微鏡イメージングおよびサポートされている脂質二重層のフォーススペクトロスコピー

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

生物工学

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

平面支持された二分子層​​上の単一分子蛍光顕微鏡

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

AFMベースの力分光法のための単一分子の共有結合

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

SLP1ポリマーと単層のAuのインタラクションから

要約

さらに動画を探す

この動画の章

その場 SIMSおよび質量選択されたイオンのソフトランディングで調製し明確に定義された表面の赤外分光法における

原子間力分光法による単一分子の接着性を調査

動的タンパク質複合体の分析の組み立てし、質量分析法と電子顕微鏡を用いた Biolayer 干渉法によるバイオセンサーから解放

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

単一分子力分光法を用いて無機材料とアミノ酸とペプチドの相互作用への洞察

OaAEP1-媒介酵素合成と単分子力分光法のための重合タンパク質の固定化

原子間力顕微鏡イメージングおよびサポートされている脂質二重層のフォーススペクトロスコピー

平面支持された二分子層上の単一分子蛍光顕微鏡

AFMベースの力分光法のための単一分子の共有結合

サポートされている脂質二重層中のリガンドナノクラスター配列

SLP1ポリマーと単層のAuのインタラクションから

要約

さらに動画を探す

この動画の章

その場 SIMSおよび質量選択されたイオンのソフトランディングで調製し明確に定義された表面の赤外分光法における

原子間力分光法による単一分子の接着性を調査

動的タンパク質複合体の分析の組み立てし、質量分析法と電子顕微鏡を用いた Biolayer 干渉法によるバイオ センサーから解放

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

単一分子力分光法を用いて無機材料とアミノ酸とペプチドの相互作用への洞察

OaAEP1-媒介酵素合成と単分子力分光法のための重合タンパク質の固定化

原子間力顕微鏡イメージングおよびサポートされている脂質二重層のフォーススペクトロスコピー

平面支持された二分子層​​上の単一分子蛍光顕微鏡

AFMベースの力分光法のための単一分子の共有結合

サポートされている脂質二重層中のリガンドナノクラスター配列

動的タンパク質複合体の分析の組み立てし、質量分析法と電子顕微鏡を用いた Biolayer 干渉法によるバイオセンサーから解放

平面支持された二分子層上の単一分子蛍光顕微鏡