Die vorliegende Arbeit wurde teilweise durch die IGF-Projekt &quot;S-Sieve&quot; (490 ZBG / 1) durch das BMWi und des BMBF-Projektes &quot;Aptasens&quot; (BMBF / DLR 01RB0805A) finanziert finanziert. Besonderen Dank an Tobias J. Günther für seine wertvolle Hilfe bei der AFM-Studien und um Erik V. Johnstone zum Lesen des Manuskripts als englischer Muttersprachler. Ferner würde der Autor dieses Papieres gerne Aline Ritter und Sabrina Gurlit (vom Institut für Ressourcenökologie zur Unterstützung bei der ICP-MS-Messungen) zu danken, Manja Vogel, Nancy Unger, Karen E. Viacava und die Gruppe Biotechnologie des Helmholtz-Institut Freiberg für Ressourcentechnologie.

Die Autoren haben nichts zu offenbaren.

In dieser Arbeit untersuchten die Bindung von Au auf S-Layer-Proteine ​​wurde unter Verwendung einer Kombination von verschiedenen analytischen Methoden untersucht. Insbesondere die Bindung von Au ist sehr attraktiv, nicht nur für die Wiedergewinnung von Au aus dem Bergbau, Wasser oder Prozesslösungen, sondern auch für die Konstruktion von Materialien, zB sensorischen Oberflächen. Für Studien der Wechselwirkung Au (Au (III) und Au (0) -NPs) mit suspendierten und umkristallisiert Monoschicht von SLP1, d...

Aufgrund der zunehmenden Verwendung von Gold für verschiedene Anwendungen wie Elektronik, Katalysatoren, Biosensoren oder medizinische Instrumente hat sich die Nachfrage des Edelmetalls in den letzten Jahren 6-9 gewachsen. Gold sowie viele andere wertvolle und Schwermetalle in die Umwelt über industrielle Abwässer in verdünnten Konzentrationen durch Bergbauaktivitäten freigesetzt, und Entsorgungs 7,8,10, obwohl die meisten Umweltkontamination durch schwere oder Edelmetalle ist ein fortlaufender Prozess vor allem durch technologische Aktivitäten verursacht. Dies führt zu einer erheblichen Störung der natürlichen Ökosysteme und könnte möglicherweise die menschliche Gesundheit gefährden 9. Die Kenntnis dieser negativen Ergebnisse fördert die Suche nach neuen Techniken, um Metalle aus kontaminierten Ökosystemen und Verbesserungen beim Recycling von Metallen aus industriellen Abwässern zu entfernen. Etablierte physikalisch-chemische Methoden wie Fällung oder Ionenaustausch sind nicht so effektiv, vor allem in der Hochly verdünnten Lösungen 7,8,11. Biosorption entweder mit lebenden oder toten Biomasse, ist eine attraktive Alternative für die Abwasserbehandlung 10,12. Die Verwendung solcher biologischer Materialien kann der Verbrauch von toxischen Chemikalien zu verringern. Viele Mikroorganismen beschrieben worden zu sammeln oder zu immobilisieren Metallen. Zum Beispiel Zellen der Lysinibacillus sphaericus (L. sphaericus) JG-A12 haben hohe Bindungskapazitäten für Edelmetalle, dargestellt beispielsweise Pd (II), Pt (II), Au (III) und anderen toxischen Metallen wie Pb (II) oder U (VI) 4,13, Zellen des Bacillus megaterium für Cr (VI) 14, Zellen von Saccharomyces cerevisiae für Pt (II) und Pd (II) 15 und Chlorella vulgäre zur Au (III) und U (VI) 16 , 17. Die Bindung von Metallen wie Au vorherigen (III), Pd (II) und Pt (II) wurde auch für Desulfovibrio desulfuricans 18 gemeldet und L. sphaericus JG-B53 19,20. Dennoch sind nicht all Mikroben binden große Mengen von Metallen und deren Anwendung als Sorptionsmaterial begrenzt 12,21. Weiterhin Metallbindungskapazität hängt von verschiedenen Parametern, beispielsweise Zell-Zusammensetzung, die verwendet Biokomponente oder Umwelt- und experimentellen Bedingungen (pH, Ionenstärke, Temperatur etc.). Das Studium der isolierten Zellwandfragmente 22,23, wie Membranlipiden, Peptidoglycan, Proteinen oder anderen Komponenten kann dabei die Metallbindungsverfahren komplexer aufgebaut ganzen Zellen 8,21 zu verstehen. Die Zellbestandteile in dieser Studie konzentrierte sich auf sind S-Layer-Proteine. S-Layer-Proteine ​​sind Teile der äußeren Zellhülle von vielen Bakterien und Archaebakterien, und sie machen etwa 15 bis 20% der Gesamtproteinmasse dieser Organismen. Wie die erste Schnittstelle für die Umwelt, diese Zell Verbindungen beeinflussen die Bakterien Sorptionseigenschaften 3. S-Schicht-Proteine ​​mit Molekulargewichten im Bereich von vierzigHunderte von kDa sind innerhalb der Zelle produziert, aber außerhalb montiert, wo sie in der Lage, Schichten auf den Lipidmembranen oder polymere Zellwandbestandteile bilden. Nach der Isolierung fast alle S-Layer-Proteine ​​die intrinsische Eigenschaft, sich spontan durch Selbstorganisation in Suspension, an Grenzflächen oder auf Oberflächen bilden ebene oder röhrenförmige Gebilde 3. Die Dicke der Proteinmonoschicht hängt von den Bakterien und ist in einem Bereich von 5 bis 25 nm 24. Im Allgemeinen können die gebildeten S-Layer-Protein-Strukturen eine schräge (P1 oder P2), quadratisch (p4) oder hexagonal (p3 oder P6) symmetrisch Gitterkonstanten von 2,5 bis 35 nm 3,24 haben. Die Gitterbildung scheint in vielen Fällen abhängig von zweiwertigen Kationen und hauptsächlich Ca 2+ 25,26, Raff, J. et al. S-Layer-basierte Nanokomposite für industrielle Anwendungen in Proteinbasis Engineered Nanostrukturen. (Hrsg Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (eingereicht)). Dennoch ist die vollständige Reaktionskaskade des Monomers Faltung Monomer-Monomer-Wechselwirkung, die Bildung eines Gitters, und die Rolle, die aus unterschiedlichen Metallen, insbesondere zweiwertigen Kationen, wie Ca 2+ und Mg 2+, noch nicht vollständig verstanden. Die Gram-positive Stamm L. sphaericus JG-B53 27 (von Bacillus sphaericus, nachdem neue phylogenetische Einordnung umbenannt) wurde aus dem Uranbergbau Halde &quot;Haberland&quot; (Johanngeorgenstadt, Sachsen, Deutschland) 4,28,29 isoliert. Seine Funktions S-Layer-Protein (SLP1) einen quadratischen Gitter, ein Molekulargewicht von 116 kDa 30 und eine Dicke von ≈ 10 nm an lebenden Bakterienzellen 31. In früheren Studien wurde die in vitro Bildung einer geschlossenen und stabilen Proteinschicht mit einer Dicke von etwa 10 nm in weniger als 10 min 19 erreicht. Die verwandten Stammes L. sphaericus JG- angezeigtA12, auch ein Isolat aus dem &quot;Haberland&quot; Haufen, besitzt eine hohe Metallbindungskapazitäten und seiner isolierten S-Schicht-Protein hat eine hohe chemische und mechanische Stabilität und gute Sorptionsraten für Edelmetalle wie Au (III) gezeigt, Pt (II), und Pd (II) 4,32,33. Diese Bindung von Edelmetallen ist mehr oder weniger spezifisch für bestimmte Metalle und hängt von der Verfügbarkeit der funktionellen Gruppen auf der äußeren und inneren Proteinoberfläche des Polymers und die in ihren Poren, der Ionenstärke und dem pH-Wert. Relevante funktionelle Gruppen für die Metall-Wechselwirkung durch die Proteine ​​sind COOH-, NH 2 -, OH-, PO 4 -, SO 4 - und SO. Grundsätzlich Metallbindungskapazitäten eröffnen ein breites Spektrum von Anwendungen, Raff, J. et al. S-Layer-basierte Nanokomposite für industrielle Anwendungen in Proteinbasis Engineered Nanostrukturen. (Hrsg Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (eingereicht)). zB als biosorptive Komponenten zur Entfernung oder Wiederherstellungvon gelösten toxischen oder wertvolle Metalle, Vorlagen für die Synthese oder definierte Abscheidung von regelmäßig strukturierten metallischen Nanopartikeln (NPs) für die Katalyse und andere Bio-engineered Materialien wie Bio-sensorischen Schichten 3,5,18,33. Regelmäßig angeordnet NP Arrays wie Au (0) -NPs könnte für große Anwendungen, die von der molekularen Elektronik und Biosensoren, Ultrahochdichte-Speichergeräte, und Katalysatoren für die CO-Oxidation 34-37 verwendet werden. Die Entwicklung solcher Anwendungen und intelligente Design dieser Materialien erfordert ein tieferes Verständnis der zugrundeliegenden Metallbindungsmechanismen. Eine Voraussetzung für die Entwicklung solcher Materialien auf biologischer Basis ist die zuverlässige Ausführung eines Schnittstellenschicht zwischen dem Biomolekül und der technische Oberfläche 38,39. Zum Beispiel Polyelektrolyte mit der Schicht-für-Schicht (LBL) Technik 40,41 zusammengebaut wurden als Zwischenschicht zur Rekristallisation von S-Layer-Proteine ​​39 verwendet wurden </sup&gt;. Eine solche Schnittstelle bietet eine relativ einfache Möglichkeit, die Proteinbeschichtung auf eine reproduzierbare und quantitative Weise auszuführen. Durch Durchführung verschiedener Experimente mit und ohne Modifikation mit Klebevermittler, ist es möglich, Aussagen über die Beschichtungs Kinetik, Schichtstabilität und die Interaktion von Metallen mit Biomolekülen 19,42, Raff, J. et al machen. S-Layer-basierte Nanokomposite für industrielle Anwendungen in Proteinbasis Engineered Nanostrukturen. (Hrsg Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (eingereicht)). Jedoch ist die komplexen Mechanismen der Proteinadsorption und Protein-Oberflächen-Wechselwirkung nicht vollständig verstanden. Vor allem Informationen über Konformation, Muster Orientierung und Beschichtungsdichten fehlt noch. Quarzmikrowaage mit Verlustüberwachung (QCM-D) Technik hat sich die Aufmerksamkeit in den letzten Jahren als ein Werkzeug für die Untersuchung der Proteinadsorption, Beschichtungs Kinetik und Interaktion pro zogenProzesse auf der Nanometerskala 19,43-45. Diese Technik ermöglicht die detaillierte Erfassung der Masse Adsorption in Echtzeit und kann als Indikator für das Protein selbst Montage und die Kopplung der funktionellen Moleküle auf Proteingitter 19,20,42,46-48 verwendet werden. Darüber hinaus öffnen QCM-D-Messungen die Möglichkeit, Metallwechselwirkungsprozesse mit der proteinhaltigen Schicht unter natürlichen biologischen Bedingungen zu studieren. In einer neueren Studie der Wechselwirkung des S-Layer-Protein mit ausgewählten Metallen wie Eu (III), Au (III), Pd (II) und Pt (II) wurde mit der QCM-D 19,20 sucht. Die adsorbierte Proteinschicht kann als vereinfachtes Modell eines Zellwand von gram-positiven Bakterien dienen. Die Untersuchung dieses einzelne Komponente kann zu einem tieferen Verständnis der Metall-Wechselwirkung beitragen. Allerdings wissen nur QCM-D-Experimente nicht Aussagen in Bezug auf Oberflächenstrukturen und Einflüsse von Metallen, um Protein zu ermöglichen. Andere Techniken sind notwendig, um diese Informationen zu erhalten. Eine postung für Abbildungs ​​bio-Nanostrukturen und Gewinnung von Informationen über Struktureigenschaften ist die Rasterkraftmikroskopie (AFM). Das Ziel der vorliegenden Studie war es, die Sorption von Gold (Au (III) und Au (0) -NPs) bis S-Layer-Proteine ​​zu untersuchen, insbesondere SLP1 von L. sphaericus JG-B53. 5.0 Verwendung von ICP-MS und immobilisiert S-Schichten unter Verwendung von QCM-D - Experimente wurden mit suspendierten Proteine ​​auf Ansatzgröße in einem pH-Bereich von 2,0 durchgeführt. Zusätzlich wurde der Einfluß der Metallsalzlösung auf dem Gitter Stabilität mit anschließender AFM-Studien untersucht. Die Kombination dieser Techniken trägt zu einem besseren Verständnis der in-vitro-Metall-Interaktionsprozesse als Werkzeug für das Lernen mehr über Bindungsereignisse auf ganze Bakterienzellen in Bezug auf bestimmte Metall Affinitäten. Diese Erkenntnis ist nicht nur wichtig für die Entwicklung von anwendbaren Filtermaterialien zur Rückgewinnung von Metallen für den Umweltschutz und zur Erhaltung der WiederQuellen 49, sondern auch für die Entwicklung von hoch geordneten Arrays von metallischen Nanopartikeln für verschiedene technische Anwendungen.

<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.

Kultivierung von Mikroorganismen und SLP1 Charakterisierung Die aufgezeichneten Daten des Bakterienwachstums zeigt das Ende der exponentiellen Wachstumsphase bei etwa 5 Stunden. Frühere Untersuchungen haben gezeigt, dass SLP1 können von diesem Zeitpunkt der Ernte (4,36 g / L nasser Biomasse (≈ 1,45 g / L (BDW)) mit einer maximalen Ausbeute 19 isoliert werden. Dennoch Optimierung der Kultivierung...

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.

Au-Wechselwirkung von SLP1 Polymere und Monolayer aus<em&gt; Lysinibacillus sphaericus</em&gt; JG-B53 - QCM-D, ICP-MS und AFM als Werkzeuge für Biomolekül-Metallforschung

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

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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 Aufrufe.  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.

Serie: 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.

In-situ-SIMS und IR-Spektroskopie von gut definierten Oberflächen durch weiche Landung der Messe-Ionen ausgewählt Vorbereitet

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

Forschung

Chemie

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.

Untersuchung von Einzelmolekül Haftung durch Rasterkraftspektroskopie

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

Ingenieurwesen

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.

Analyse dynamischer Proteinkomplexe montiert auf und befreit Biolayer Interferometrie Biosensor mit Massenspektrometrie und Elektronenmikroskopie

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

Biochemie

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.

Dissipative Microgravimetry die Bindung Dynamik der Phospholipid-bindende Protein Annexin A2, solide unterstützt Lipid Bilayer mit einem Quarz-Resonator zu studieren

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.

Einblicke in die Wechselwirkungen von Aminosäuren und Peptide mit anorganischen Materialien Verwendung von Einzelmolekülkraftspektroskopie

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-Vermittelte enzymatische Synthese und Immobilisierung von polymerisiertem Protein für die Einzelmolekül-Kraftspektroskopie

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

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.

Einzelmolekülfluoreszenzmikroskopie auf Planar Unterstützte 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 ...

Biotechnik

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.

Kovalente Befestigung einzelner Moleküle für die AFM-basierte Kraftspektroskopie

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.

Ligand Nano-Cluster-Arrays in einem unterstützten Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-Chip: Eine Label-freie Studie von Protein-Phosphoinositid-Wechselwirkungen

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Au-Wechselwirkung von SLP1 Polymere und Monolayer aus

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