Name: au-interaction of slp1 polymers and monolayer from lysinibacillus sphaericus jg-b53 - qcm-d, icp-ms and afm as tools for biomolecule-metal studies
Uploaded: 2016-01-19T14:00:00.000+00:00
Duration: 8 min 29 s
Description: 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

Bu çalışma kısmen BMWi ve BMBF-proje &quot;Aptasens&quot; (BMBF / DLR 01RB0805A) tarafından finanse edilen IGF-projesi &quot;S-Elek&quot; (490 ZBG / 1) tarafından finanse edildi. AFM çalışmaları sırasında ve ana dili İngilizce olan konuşmacı olarak el yazması okumak için Erik V. Johnstone yaptığı değerli yardım için Tobias J. Günther Special thanks. Dahası, bu makalenin yazarı (ICP-MS ölçümlerde yardım için kaynak Ekoloji Enstitüsü) Aline Ritter ve Sabrina Gurlit teşekkür etmek istiyorum, Manja Vogel, Nancy Unger, Karen E. Viacava ve Helmholtz-Enstitüsü grup biyoteknoloji Kaynak Teknoloji Freiberg.

Yazarlar ifşa hiçbir şey yok.

Bu çalışmada farklı analiz yöntemleri bir arada kullanarak araştırıldı S katmanlı proteinlere bağlanmasının Au incelenmiştir. Özellikle, Au bağlayıcı maden suları veya işlem çözeltilerinden Au geri kazanımı için değil, aynı zamanda malzeme, örneğin, duyu yüzeylerin inşası için de oldukça ilgi çekicidir. Au etkileşim çalışmaları için (Au (III) ve Au (0) -NPs) süspansiyon haline getirilmiş ve Slp1 tek tabaka yeniden kristalize olan protein sahip izole edilmesi. Bu nedenl...

Nedeniyle elektronik, katalizörler, biyosensörler, ya da tıbbi cihazlar gibi birçok uygulama için altın artan kullanımı, bu değerli metalin talebi son birkaç yıllık süre 6-9 üzerinde büyüdü. Ağır veya kıymetli madenlerin en çevresel kirlenme devam eden bir süreç olmasına rağmen Altın yanı sıra diğer birçok değerli ve ağır metaller, madencilik faaliyetleri ile, seyreltik konsantrasyonlarda endüstriyel atık yoluyla çevreye salınan ve atık 7,8,10 edilir özellikle teknolojik faaliyetlerinden kaynaklanan. Bu doğal ekosistemlerin önemli bir girişime yol açar ve potansiyel olarak insan sağlığı 9 tehdit edebilir. Bu olumsuz sonuçların bilinmesi yeni teknikler endüstriyel atık sulardan metallerin geri dönüşümü kontamine ekosistemler ve iyileştirmeler metallerin kaldırmak için arama teşvik etmektedir. Çökeltme veya iyon değişimi gibi köklü fiziko-kimyasal yöntemler, özellikle yüksek olarak, çok etkili değildirly çözümler 7,8,11 seyreltilmiştir. Biyosorpsiyon ya yaşayan ya da ölü biyokütle ile, atıksu arıtımı 10,12 cazip bir alternatif. Bu tür biyolojik malzemelerin kullanımı toksik kimyasalların tüketimini azaltabilirsiniz. Birçok mikroorganizmalar birikebilir ya da metalleri hareketsiz kılınması için tarif edilmiştir. Örneğin, Lysinibacillus spaericus&#39;dan hücreleri (L. sphaericus) JG-A12 değerli metaller için yüksek bağlanma kapasiteleri göstermişlerdir, örneğin, Pd (II), Pt (II), Au (III), ve Pb gibi diğer toksik metaller (II) ya da U (VI) &#39;4,13, Cr (VI) 14 Bacillus megaterium hücreleri, Au, Pt (II) ve Pd (II), 15 ve Chlorella vulgar için Saccharomyces cerevisiae hücreleri (III) ve U (VI)&#39; ya 16 , 17. Au gibi önceki metallerin bağlanması (III), (II) Desulfovibrio rapor edilmiştir Pd (II), ve Pt 18 ve L. Desulfovibrio sphaericus JG-B53 19,20. Bununla birlikte, All mikroplar metallerin yüksek miktarda bağlama ve emmeyle malzeme olarak uygulama sınırlı 12,21 olduğunu. Ayrıca, bağlama kapasitesi, metal, örneğin, farklı parametreler, cep bileşimi olup, kullanılan biyo-bileşenli veya çevresel ve deneysel koşullar (pH, iyonik kuvvet, sıcaklık vs.) bağlıdır. Izole edilmiş hücre duvarı parçaları 22,23 arasında çalışması, membran lipidleri, peptidoglikan, proteinler veya diğer bileşenler gibi karmaşık yapılan bütün hücrelerin 8,21 süreçlerini bağlama metal anlamaya yardımcı olur. Bu çalışmada odaklanmış hücre bileşenleri S katmanlı proteinlerdir. S-katmanlı proteinleri birçok bakteri ve arkelerin dış hücre zarfın parçaları vardır ve bunlar yaklaşık 15 oluşturmaktadır - bu organizmaların toplam protein kütlesinin% 20. Çevreye ilk arayüz olarak, bu hücre bileşikler güçlü bakteriyel emme özellikleri 3 etkilemektedir. Moleküler ağırlıklar kırk kadar S-tabaka proteinlerikDa&#39;lık yüz hücre içinde üretilen, ancak dış tarafına monte edilmiş onlar lipid zarlar ya da polimerik hücre duvarı bileşenleri ile ilgili tabakaları oluşturmak mümkün olduğu. Izole sonra, hemen hemen tüm S katmanlı proteinleri kendiliğinden ara yüzlerde ya da düzlemsel ya da tüp benzeri yapılar 3 oluşturma yüzeyleri üzerine, süspansiyon içinde kendini düzenleyen içsel bir özelliği vardır. Protein tek tabaka kalınlığı bakteriler bağlıdır ve 5 aralığında olan - 25 nm 24. Genel olarak, oluşan S-katmanlı protein yapıları 35 nm 3,24 bir verev (p1 veya p2), kare (p4), ya da altıgen (p3 veya p6) 2.5 kafes sabitleri ile simetri olabilir. Kafes oluşumu, iki değerli katyonları bağlıdır ve esas olarak Ca2 + 25,26, Raff, J. ve diğerleri birçok durumda olduğu görülmektedir. S-katmanlı Protein temelli Engineered Nanoyapıların endüstriyel uygulamalar için esaslı nanokompozitler. (eds Tijana Z. Grove ve Aitziber L. Cortajarena) (Springer, 2016 (gönderilen)). Bununla birlikte, özellikle Ca2 + ve Mg2 + gibi divalent katyonlar monomer katlama, monomer etkileşimi, bir kafes oluşması ve farklı metaller rolünün tam tepkime zincirine, hala tam olarak anlaşılmamıştır. Gram-pozitif suş L. 27 (yeni filogenetik sınıflandırmaya sonra Bacillus spaericus&#39;dan yeniden adlandırılmış) sphaericus JG-B53 uranyum madenciliği atık yığını &quot;Haberland&quot; (Johanngeorgenstadt, Saksonya, Almanya) 4,28,29 izole edilmiştir. Fonksiyonel S katmanlı proteini (Slp1) kare kafes, 116 kDa&#39;lık 30 kadar bir molekül ağırlığına, ve bakteri hücreleri 31 yaşamaya ≈ 10 nm&#39;lik bir kalınlığa sahiptir. Daha önce yapılan çalışmalarda, yaklaşık 10 nm kalınlığında bir kapalı ve istikrarlı bir protein tabakasının in vitro oluşumunu en az 10 dakika 19 elde edildi. Ilgili suş L. sphaericus JG-A12, aynı zamanda &quot;Haberland&quot; yığın bir izolat, daha yüksek metal bağlanma kapasitesine sahip olan ve onun izole edilmiş bir S-tabaka proteini Au gibi değerli metaller için yüksek bir kimyasal ve mekanik kararlılık ve iyi emme oranları göstermiştir (III), Pt (II), ve Pd (II) 4,32,33. Değerli metallerin bağlantılarının çok veya daha az spesifik bir metal olduğu ve polimerin iç ve dış yüzeyi üzerinde, protein ve gözeneklerde fonksiyonel gruplar, iyonik kuvvet kullanılabilirliği ve pH değerine göre değişir. , OH-, PO 4 - - SO 4 - ve SO proteinler tarafından metal etkileşimi için, ilgili fonksiyonel gruplar COOH-, NH 2 bulunmaktadır. Prensip olarak, metal bağlanma kapasitesi uygulamalarda, Raff, J. ve diğerleri, geniş bir spektrum açın. S-katmanlı Protein temelli Engineered Nanoyapıların endüstriyel uygulamalar için esaslı nanokompozitler. (editörler Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (gönderilen)). Örneğin, olarak kaldırılması veya geri kazanımı için biosorptive bileşenlerçözünmüş toksik veya değerli metallerin, sentez veya düzenli yapılandırılmış metalik nanopartiküller (NPS) kataliz için, ve biyo-duyusal katmanlar 3,5,18,33 gibi diğer biyo-mühendislik malzemeleri tanımlı birikimi için şablonlar. Au gibi düzenli düzenlenmiş NP diziler (0) -NPs CO-oksidasyonu 34-37 moleküler elektronik ve biyosensörlere, ultrahigh yoğunluk depolama aygıtları ve katalizörler kadar önemli uygulamalar için kullanılabilir. Bu tür uygulamalar ve bu malzemelerin akıllı tasarım geliştirme altında yatan metal bağlama mekanizmaları daha derin bir anlayış gerektirmektedir. Bu tür biyolojik bazlı malzemeler geliştirilmesi için bir ön koşuldur biyomolekül ve teknik yüzey 38,39 arasında bir ara-yüz tabakası güvenilir bir uygulamasıdır. Örneğin, polielektrolitler, S-tabaka proteinleri 39 arasında yeniden kristalizasyon için bir ara-yüz tabakası olarak kullanılmaktadır katman-katman (Lbl) tekniği 40,41 monte </&gt; sup. Böyle bir arayüz, bir tekrar üretilebilir ve niceliksel bir şekilde protein bir kaplama yapılması için nispeten kolay bir yol sunar. Ve yapışkan promotörler ile değişiklik olmadan, farklı deneyler ile, kaplamanın kinetik, tabaka stabilitesini ve biyomoleküllerin 19,42, Raff, J. ve ark metallerin etkileşimi ile ilgili açıklama yapmak mümkündür. S-katmanlı Protein temelli Engineered Nanoyapıların endüstriyel uygulamalar için esaslı nanokompozitler. (editörler Tijana Z. Grove &amp; Aitziber L. Cortajarena) (Springer, 2016 (gönderilen)). Bununla birlikte, protein adsorpsiyonu ve proteinsiz yüzey etkileşimi kompleks mekanizması tam olarak anlaşılmış değildir. Özellikle konformasyon, desen yönü ve kaplama yoğunlukları hakkında bilgi hala kayıp. Dağıtma izlenmesi (QCM-D) tekniği ile Kuvars kristal mikroterazi bir protein adsorpsiyon, kaplama kinetik eğitim için bir araç, ve etkileşim yanlısı olarak son yıllarda dikkat çekennanometre ölçeğinde 19,43-45 üzerine süreçlerle. Bu teknik, gerçek zamanlı olarak kütle adsorpsiyon detaylı bir şekilde tespit eder ve protein örgüleri 19,20,42,46-48 protein kendinden montaj işlemi ve fonksiyonel moleküllerin bağlanması için bir gösterge olarak kullanılabilir. Buna ek olarak, QCM-D ölçümleri doğal biyolojik şartlarda proteinli tabaka ile metal etkileşim süreçlerini incelemek için olasılığını açık. Yeni bir çalışmada, Eu gibi seçilen metaller ile S-katmanlı protein etkileşimi (III), Au (III), Pd (II) ve Pt (II) QCM-D 19,20 ile ele alınmıştır. Emilmiş olan protein tabakası, gram-pozitif bakterilerin hücre duvarı basitleştirilmiş bir model olarak hizmet edebilir. Bu tek bileşenli çalışma metal etkileşimi daha derin bir anlayış katkıda bulunabilir. Ancak, sadece QCM-D deneyleri yüzey yapıları ve proteine ​​metallerin etkileri ile ilgili ifadeleri izin vermez. Diğer teknikler, bilgi elde etmek için gereklidir. Bir posyapısal özelliklerine görüntüleme biyo-nanoyapılarda için gündeme gelince ve bilgi alma atomik kuvvet mikroskopisi (AFM) &#39;dir. Bu çalışmada elde edilen amacı, L., özellikle Slp1 içinde, ((0) -NPs Au (III) ve Au) S-tabaka proteinleri altın emilimini araştırmaktır sphaericus JG-B53. ICP-MS kullanılarak 5.0 ve QCM-D kullanılarak immobilize S-tabakaları ile - Deneyler 2.0 arasında bir pH aralığı içinde toplu ölçekte süspansiyon protein ile yapıldı. Buna ek olarak, kafes stabilitesi üzerindeki metal tuzu çözeltisinin etkisi takip eden AFM çalışmalarla araştırılmıştır. Bu tekniklerin kombinasyonu belirli metal afinite ile ilgili tüm bakteri hücreleri olayları bağlayıcı hakkında daha fazla öğrenme için bir araç olarak in vitro metal etkileşim süreçlerinin daha iyi anlaşılmasına katkıda bulunur. Bu bilgi, sadece çevre koruma metallerin geri kazanımı için uygun filtre malzemeleri geliştirilmesi ve yeniden korunması için önemlidir49, aynı zamanda çeşitli teknik uygulamalar için çok düzenli metal NP dizilerinin geliştirilmesi için kaynaklar.

<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.

Mikroorganizmalar ve Slp1 karakterizasyonu Yetiştirme Bakteri büyümesinin kaydedilen veriler yaklaşık 5 saat sonra üslü büyüme fazında sonunu gösterir. Önceki araştırmalar Slp1 hasat Bu noktada (4,36 g / L, ıslak biyo-kütle (≈ 1,45 g / L (BDW)), maksimum verimle 19 ile izole edilebilir olduğunu göstermiştir. Bununla birlikte, tanımlanmış ortam bileşenleri kullanarak veya yet...

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 Polimerler ve Monolayer Au-etkileşimden<em&gt; Lysinibacillus sphaericus</em&gt; JG-B53 - QCM-D, biyomoleküler metal Çalışmaları Araçlar olarak ICP-MS ve 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 San Sebastian

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 Görüntüleme.  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.

Video: 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.

Kütle-Seçilen İyonlar Yumuşak İniş Hazırlayan İyi tanımlanmış Yüzeyler Yerinde SIMS ve IR spektroskopisi

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

Kimya

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.

Atomik Kuvvet Spektroskopi Tek Molekül Yapışma incelenmesi

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

Mühendislik

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.

Dinamik Protein kompleksleri analiz monte ve kütle spektrometresi ve Elektron Mikroskopisi kullanılarak Biolayer Interferometry biyoalgılayıcı--dan serbest bırakmak

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

Biyokimya

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.

A2 bir kuvars rezonatör kullanarak katı destekli Lipid Bilayers için Annexin Protein bağlayıcı fosfolipid bağlama dinamikleri çalışmaya dissipative Microgravimetry

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.

Tek Molekül Kuvvetleri Spektroskopisi kullanarak İnorganik Malzemelerle Amino Asitler ve Peptitler Etkileşimi içine Trendleri

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-Tek Moleküllü Kuvvet Spektroskopisi İçin Polimerize Proteinin Aracılı Enzimatik Sentezi ve Immobilizasyonu

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.

Atomik Kuvvet Mikroskobu Görüntüleme ve Desteklenen Lipid Bilayers Kuvvet Spektroskopisi

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

Biyomühendislik

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.

Planar on Tek Molekül Floresans Mikroskopi bilayers Desteklenen

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 tabanlı Kuvvet Spektroskopisi için Tek Moleküllerin Kovalent Eki

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 Polimerler ve Monolayer Au-etkileşimden

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