O presente trabalho foi parcialmente financiado pelo IGF-projeto &quot;S-Peneira&quot; (490 ZBG / 1) financiado pela BMWi eo BMBF-projeto &quot;Aptasens&quot; (BMBF / DLR 01RB0805A). Um agradecimento especial a Tobias J. Günther pelo valioso auxílio durante os estudos de AFM e para Erik V. Johnstone para ler o manuscrito como um falante nativo Inglês. Além disso, o autor deste papel gostaria de agradecer Aline Ritter e Sabrina Gurlit (do Instituto de Ecologia de Recursos para assistência em medições por ICP-MS), Manja Vogel, Nancy Unger, Karen E. Viacava e da biotecnologia grupo do Instituto Helmholtz- Freiberg para a Tecnologia de Recursos.

Os autores não têm nada a revelar.

Neste trabalho foi estudada a ligação às proteínas de Au-S camada foi investigada utilizando uma combinação de diferentes métodos analíticos. Em particular, a ligação de Au é muito atraente, não somente para a recuperação das águas de mineração de Au ou soluções de processo, mas também para a construção de materiais, por exemplo, superfícies sensoriais. Para os estudos da interacção Au (Au (III) e Au (0) -NPs) com suspensão e recristalizou-se em monocamada de Slp1, a proteína...

Devido ao crescente uso de ouro para diversas aplicações como eletrônica, catalisadores, biossensores, ou instrumentos médicos, a procura deste metal precioso tem crescido ao longo dos últimos anos de tempo 6-9. Ouro, bem como muitos outros metais preciosos e pesados ​​são liberados no ambiente através de efluentes industriais em concentrações diluídas, por meio de atividades de mineração, e eliminação de resíduos 7,8,10, embora a maioria contaminação ambiental por metais pesados ​​ou preciosos é um processo em curso causada principalmente por atividades tecnológicas. Isto leva a uma interferência significativa dos ecossistemas naturais e pode potencialmente ameaçar a saúde humana 9. Conhecendo esses resultados negativos promove a busca por novas técnicas para remover metais de ecossistemas e melhorias contaminados na reciclagem de metais de efluentes industriais. Métodos físico-químicos bem estabelecidos como precipitação ou permuta de iões não são tão eficazes, especialmente em altaly diluída soluções 7,8,11. Biossorção, quer com a vida ou biomassa morta, é uma alternativa atraente para o tratamento de águas residuais 10,12. O uso de tais materiais biológicos pode reduzir o consumo de produtos químicos tóxicos. Muitos microrganismos têm sido descritos para acumular ou imobilizar metais. Por exemplo, células de Lysinibacillus sphaericus (L. sphaericus) JG-A12 mostraram capacidades de ligação elevadas para os metais preciosos, por exemplo, Pd (II), Pt (II), Au (III), e outros metais tóxicos como o Pb (II) ou L (VI) 4,13, células de Bacillus megaterium para o Cr (VI), 14, células de Saccharomyces cerevisiae para Pt (II) e Pd (II) 15, e Chlorella vulgar para Au (III) e U (VI) 16 , 17. A ligação de metais anteriores como Au (III), Pd (II) e Pt (II), também tem sido relatado para Desulfovibrio desulfuricans 18 e para L. sphaericus JG-B53 19,20. No entanto, não all micróbios vincular quantidades elevadas de metais e sua aplicação como material de sorção é limitada 12,21. Além disso, a capacidade de ligação do metal depende de diferentes parâmetros, por exemplo, a composição celular, o bio-componente usado, ou condições ambientais e experimentais (pH, força iónica, temperatura, etc.). O estudo dos fragmentos da parede celular isolado 22,23, como lípidos da membrana, peptidoglycan, proteínas ou outros componentes, ajuda a compreender os processos de células inteiras construídas complexas 8,21 vinculativo metal. Os componentes celulares com foco em neste estudo são proteínas S-layer. Proteínas S-camada são partes do envelope celular externo de muitas bactérias e archaea, e que constituem cerca de 15 - 20% da massa de proteína total destes organismos. À medida que a primeira interface para o meio ambiente, estes compostos celulares influenciam fortemente as propriedades de sorção bacterianas 3. Proteínas S-layer com pesos moleculares variando de quarentaa centenas de kDa são produzidos dentro da célula, mas são montados no exterior, onde eles são capazes de formar camadas nas membranas lipídicas ou poliméricas componentes da parede celular. Uma vez isolado, quase todos os S-layer proteínas têm a propriedade intrínseca à espontaneamente auto-montar em suspensão, nas interfaces, ou em superfícies planas ou formando tubo-como estruturas 3. A espessura da monocamada de proteína depende das bactérias e está dentro de uma gama de 5 - 24 25 nm. Em geral, as estruturas de proteínas S-camada formada pode ter uma oblíqua (P1 ou P2), quadrada (P4) ou hexagonal (P3 ou P6) simetria com constantes de rede de 2,5 a 35 nm, 3,24. A formação da estrutura parece ser, em muitos casos dependentes de catiões divalentes e principalmente sobre Ca 2+ 25,26, Raff, J. et ai. S-camada de nanocompósitos à base para aplicações industriais em à base de proteínas Engineered Nanoestruturas. (eds Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (submetido)). No entanto, a cascata de reacção cheio de dobragem monómero, monómero interação-monómero, a formação de uma estrutura, e o papel dos diferentes metais, especialmente de catiões divalentes, tais como Ca 2+ e Mg 2+, não são ainda totalmente compreendidas. A estirpe de bactérias gram-positivas L. sphaericus JG-B53 (renomeado de Bacillus sphaericus após nova classificação filogenética) 27 foi isolada dos resíduos pilha mineração de urânio &quot;Haberland&quot; (Johanngeorgenstadt, Saxônia, Alemanha) 4,28,29. Sua funcional da proteína S-camada (Slp1) possui uma rede quadrada, um peso molecular de 116 kDa, 30, e uma espessura de 10 nm ≈ em células de bactérias que vive 31. Em estudos anteriores, a formação in vitro de uma camada de proteína fechado e estável com uma espessura de aproximadamente 10 nm foi alcançada em menos de 10 min 19. A tensão relacionada L. sphaericus JG-A12, também um isolado da pilha &quot;Haberland&quot;, possui capacidades de ligação de alta de metal e seu isolado S-camada de proteína tem mostrado uma boa taxas de sorção de alta estabilidade química e mecânica e para os metais preciosos como Au (III), Pt (II), e Pd (II) 4,32,33. Esta ligação de metais preciosos é mais ou menos específicos para alguns metais e depende da disponibilidade de grupos funcionais sobre a superfície da proteína exterior e interior do polímero e nos seus poros, força iónica, e o valor de pH. Grupos funcionais relevantes para a interação do metal por as proteínas são COOH-, NH 2 -, OH-, PO 4 -, SO 4 -, e SO. Em princípio, as capacidades de ligação a metais abrir um amplo espectro de aplicações, Raff, J. et ai. S-camada de nanocompósitos à base para aplicações industriais em à base de proteínas Engineered Nanoestruturas. (eds Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (submetido)). por exemplo, como componentes biosorptive para remoção ou recuperaçãode metais tóxicos ou valiosos dissolvidos, modelos para a síntese ou a deposição de nanopartículas definido regularmente estruturados metálicos (PN) para a catálise e outros materiais bio-engenharia, como camadas de bio-sensorial 3,5,18,33. Matrizes NP regularmente organizados como Au (0) -NPs poderia ser usado para grandes aplicações que vão desde a eletrônica molecular e biossensores, dispositivos de armazenamento densidade extremamente altas, e catalisadores para o CO-oxidação 34-37. O desenvolvimento de tais aplicações e design inteligente desses materiais exige uma compreensão mais profunda dos mecanismos de ligação de metal subjacentes. Um pré-requisito para o desenvolvimento de tais materiais de base biológica é a implantação confiável de uma camada de interface entre a biomolécula ea superfície técnica 38,39. Por exemplo, polielectrólitos montado com a camada-por-camada (LbL) 40,41 técnica têm sido utilizados como uma camada de interface para a recristalização das proteínas S-camada 39 </sup&gt;. Uma tal interface proporciona uma maneira relativamente fácil de realizar o revestimento de proteína de uma forma reprodutível e quantitativa. Através da realização de diferentes experimentos com e sem modificação com promotores adesivas, é possível fazer afirmações sobre a cinética de revestimento, estabilidade camada, e interação de metais com biomoléculas 19,42, Raff, J. et al. S-camada de nanocompósitos à base para aplicações industriais em à base de proteínas Engineered Nanoestruturas. (eds Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (submetido)). No entanto, o mecanismo complexo da adsorção de proteína e interacção de proteína à superfície não é completamente compreendido. Especialmente informações sobre a conformação, a orientação padrão, e densidades de revestimento ainda está desaparecido. Quartz microbalança de cristal com monitoramento dissipação (QCM-D) técnica tem atraído atenção nos últimos anos como uma ferramenta para o estudo de adsorção de proteínas, cinética de revestimento, e interação proprocessos em escala nanométrica 19,43-45. Esta técnica permite a detecção detalhada de adsorção em massa em tempo real, e pode ser usado como um indicador para o processo de auto-montagem de proteínas e acoplamento de moléculas funcionais em redes proteicas 19,20,42,46-48. Além disso, as medições QCM-D em aberto a possibilidade de estudar os processos de interação do metal com a camada proteica em condições biológicas naturais. Em um estudo recente, a interacção da proteína S-camada com metais seleccionados tais como Eu (III), Au (III), Pd (II) e Pt (II) foi estudada com QCM-D 19,20. A camada de proteína adsorvida pode servir como um modelo simplificado de uma parede celular de bactérias gram-positivas. O estudo deste componente único pode contribuir para uma compreensão mais profunda da interação metal. No entanto, unicamente experimentos QCM-D não permitem declarações relativas a estruturas de superfície e influências de metais para a proteína. Outras técnicas são necessárias para obter essas informações. Um poslidade de imagem para bio-nano-estruturas e obtenção de informações sobre as propriedades estruturais é a microscopia de força atômica (AFM). O objectivo do estudo foi investigar apresentada a sorção de ouro (Au (III) e Au (0) -NPs) para proteínas S-camada, em particular Slp1 de L. sphaericus JG-B53. As experiências foram efectuadas com proteínas suspensos na escala em lotes numa gama de pH 2,0 - 5,0 utilizando ICP-MS e imobilizadas com S-camadas usando QCM-D. Além disso, a influência de uma solução de sal de metal sobre a estabilidade da estrutura foi investigada com estudos subsequentes AFM. A combinação destas técnicas contribui para uma melhor compreensão dos processos de interação in vitro de metal como uma ferramenta para aprender mais sobre os eventos em células bacterianas inteiras sobre afinidades de metal específicos de ligação. Este conhecimento é crucial não só para o desenvolvimento de materiais de filtro válidos para a recuperação de metais para a protecção do ambiente ea conservação dos refontes 49, mas também para o desenvolvimento de matrizes de nanopartículas metálicas altamente ordenadas para várias aplicações técnicas.

<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.

Cultivo de Microrganismos e Slp1 Caracterização Os dados gravados do crescimento bacteriano indica o final da fase de crescimento exponencial em cerca de 5 horas. Investigações anteriores demonstraram que Slp1 pode ser isolado a partir deste ponto de colheita (4,36 g / L de biomassa húmida (≈ 1,45 g / L (BDW)) com um rendimento máximo de 19. No entanto, a optimização de cultivo usando comp...

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-Interação de Slp1 Polímeros e monocamada de<em&gt; Lysinibacillus sphaericus</em&gt; JG-B53 - QCM-D, o ICP-MS e AFM como Ferramentas para Estudos biomolécula metal-

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

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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 visualizações.  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.

Vídeo: 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 e IR Espectroscopia de Superfícies bem definidos preparados pela Soft Landing de Íons Mass-selecionados

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

Química

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.

Investigando única molécula de adesão de Força Atômica 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 ...

Engenharia

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.

Analisando a dinâmica da proteína montada sobre e libertado da interferometria Biolayer Biosensor usando espectrometria de massa e de microscopia eletrônica

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

Bioquímica

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.

Microgravimetry dissipativo para estudar a dinâmica da vinculação do fosfolipídeo proteína anexina A2 para Bilayers do Lipid com suporte sólido usando um ressonador de quartzo

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.

Insights sobre as interações de aminoácidos e peptídeos com Materiais Inorgânicos Usando Single-Molecule Força Spectroscopy

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.

Síntese Enzimática Mediada oOAEP1 e Imobilização de Proteína Polimerizada para Espectroscopia de Força De Molécula Única

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.

Bicamadas única molécula de microscopia de fluorescência em Planar Apoiado

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 ...

Bioengenharia

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.

Ligação covalente de moléculas únicas para espectroscopia de força baseada em AFM

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.

Matrizes ligando Nano-fragmentação em uma bicamada lipídica Compatível

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: um estudo livre de rótulos de Interações Proteína-fosfoinositide

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

Au-Interação de Slp1 Polímeros e monocamada de

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