The present work was partially funded by the IGF-project &#34;S-Sieve&#34; (490 ZBG/1) funded by the BMWi and the BMBF-project &#34;Aptasens&#34; (BMBF/DLR 01RB0805A). Special thanks to Tobias J. G&#252;nther for his valuable help during AFM studies and to Erik V. Johnstone for reading the manuscript as a native English speaker. Further, the author of this paper would like to thank Aline Ritter and Sabrina Gurlit (from Institute for Resource Ecology for assistance in ICP-MS measurements), Manja Vogel, Nancy Unger, Karen E. Viacava and the group biotechnology of the Helmholtz-Institute Freiberg for Resource Technology.

The authors have nothing to disclose.

In this work studied the binding of Au to S-layer proteins was investigated using a combination of different analytical methods. In particular, the binding of Au is very attractive not only for the recovery of Au from mining waters or process solutions, but also for the construction of materials, e.g., sensory surfaces. For studies of the Au interaction (Au(III) and Au(0)-NPs) with suspended and recrystallized monolayer of Slp1, the protein had to be isolated. Therefore, this study has shown the successful cultivation of the gram-positive bacterial strain L. sphaericus JG-B53 and the isolation of the surface layer protein Slp1. Nevertheless, the cultivation and protein isolation remain challenging and should be optimized. A large scale production of biomass and S-layer proteins is a precondition for an industrial application of both, e.g., for the production of metal selective filter materials. Their application potential is undoubtedly high for the removal of toxic metals or the recovery of valuable metals dissolved in process water, waste water, or drainage water. Furthermore, the application potential for S-layers is even larger considering their additional potential in other bio-inspired materials, such as biosensors and catalysts.
The batch-sorption experiments of suspended Slp1 polymers indicated a high and stable binding of Au(III) within the investigated pH-range from 2.0 to 5.0. Thereby, metal removal efficiencies of up to 60% could be reached. This remarkable binding behavior can be explained by a strong interaction of Au(III) with Slp1 probably induced through interaction of carboxylic groups and nitrogen bearing groups present on the surface of the protein. This arguments could be strengthen by FTIR and EXAFS investigation of the similar strain L. sphaericus JG-A12 32,79. Also, intrinsic reducing properties of Slp1 can explain the high RE of Au(III) by reducing it to nanoparticular Au(0). It can be assumed that S-layer, as first interface of bacteria to the environment, should be mainly involved in metal binding. Within the investigated pH range, the highest metal binding capacity with 102.5 mg Au(III)/g Slp1 was achieved at pH 4.0. This binding capacity is higher than reported for other bio-components, e.g., for isolated cell wall of Bacillus subtilis (71.5 mg Au(III)/g) 86 or for biomass of Chlorella vulgaris (98.5 mg Au(III)/g) 8.
ICP-MS used for determination of the bound metal by Slp1 polymers is very sensitive method and enables the detection of smallest amounts of gold in this study. ICP-MS offers many benefits for performing trace metal determinations, e.g., easy handling system and low detection limits; in case of gold down to 0.1 - 1 ppt. This makes this method a versatile tool for investigating biosorptive processes in low concentration ranges. However, the results in this investigation were gained with suspended S-layer polymers and cannot be easily transferred to S-layer lattices recrystallized on surfaces, and therefore show the limitation of ICP-MS. For example, no direct relation of isolated protein polymers could be made to those S-layer structures on vital bacteria cells. In addition, the ICP-MS measurements do not allow the determination of the kinetic of metal sorption. Therefore, it is necessary to find methods more suitable for the investigation of the metal binding by thin immobilized protein films.
In the present study, QCM-D analyses were applied to detect the in situ formation of S-layer lattices on surfaces as well as the deposition of Au on the proteins. Therefore, QCM-D is a robust and reproducible method for the recognition of molecule adsorption and interaction processes . Additionally QCM-D is a relatively simple, cost-effective, and nonhazardous method to monitor such processes online. The method has the advantage to detect mass change with a maximum mass sensitivity in liquids of &#8776; 0.5 ng &#8729; cm-2. This enables the possibility to detect even weak interaction, e.g., of protein with dissolved metals or to measure adsorptions in low concentration ranges. The disadvantage of QCM-D is that this is not a structure imaging method that allows the visualization of e.g., protein lattices. Therefore, other techniques are needed.
The QCM-D analyses in this study were followed by AFM imaging. The combination of these methods allowed the study of the sorption kinetics and consequences of Au sorption for the coating, thus proving that they are versatile tools for investigating metal interaction of thin proteinaceous films. Further, it was shown that a reliable recrystallization of the S-layer proteins on technical supports is essential for subsequent protein interaction studies. Therefore, modifications of the surfaces using adhesion promotors are of interest. The described implementation of polyelectrolytes (ending with positively charged PEs) as intermediate layer between SiO2 surfaces and protein layer lead to an improved method for a fast protein coating. The positive effect of a positively charge polyelectrolyte layer has been described previously for e.g., immobilization of vital bacteria cells of L. sphaericus JG-B53 31. The implementation of the presented PE-layers are the most important and critical step for the later successful and reproducible recrystallization of S-layer proteins.
It could be demonstrated that for the investigation of thin layers of Slp1, QCM-D is a good method to show the mass adsorption respective to the protein recrystallization as monolayer films in real time. This could also be observed previously for the reassembly of the S-layer protein SbpA of L. sphaericus CCM2177 47. By using subsequent high-resolution AFM analyses changes of the lattice structure of the protein self-assemblies can be visualized. AFM measurements revealed the p4 symmetry of Slp1 and confirm the modelled layer thickness of Slp1 of about 10 nm. Also, the direct interaction of dissolved metal ions with the protein layer could be monitored by QCM-D proving good binding of Au(III) by the underlying protein layer. The probable formation of the smallest Au(0)-NPs from Au(III) solution inside protein pores caused by intrinsic reducing properties of the Slp1 lattice within QCM-D measurements could not been detected by subsequent AFM measurements. This could be related to the resolution limit of AFM and the experimental set up in this study. This shows the limitation of this technique and the necessity of high resolution imaging for biomolecules in the sub nanometer scale. However, a high stability of the recrystallized Slp1 layer could be deduced from the obtained QCM-results and was confirmed by AFM investigation.
In conclusion, it could be shown that Slp1 polymers have high metal binding capacities for gold within the investigated pH-range. Furthermore, the investigation reveals that the Slp1 lattice is a good matrix metal ion binding and for the immobilization of metallic nanoparticles. It could be shown that each method used in this article has the possibility to detect even small metal interactions, or in case of AFM can visualize structures in the nanoscale range. Although, it is only by the combination of these shown methods, that has allowed to improve the knowledge and understanding of the investigated proteins on a molecular level.
Mainly, QCM-D and AFM are the preferred methods of choice for future investigation of protein monolayer adsorption and their interaction with metals and functional molecules. By combining these two methods, the detection of S-layer protein adsorption processes and surface imaging provides an insight into molecular processes that may be able to be transferred to enhance the knowledge of living bacteria and the interaction with their environment. This study showed an excerpt of possible methods helpful for understanding protein and metal interaction. Other helpful techniques that could enhance the knowledge of such processes in detail ranging from diverse spectrometric and chromatographic methods to spectroscopic investigation of biomolecules and should be included in future studies.

Due to the increasing use of gold for several applications like electronics, catalysts, biosensors, or medical instruments, the demand of this precious metal has grown over the last few years&#39; time 6-9. Gold as well as many other precious and heavy metals are released into the environment via industrial effluents in dilute concentrations, through mining activities, and waste disposal 7,8,10,although most environmental contamination by heavy or precious metals is an on-going process mainly caused by technological activities. This leads to a significant interference of natural ecosystems and could potentially threaten human health 9. Knowing these negative outcomes promotes the search for new techniques to remove metals from contaminated ecosystems and improvements in recycling metals from industrial wastewater. Well-established physico-chemical methods like precipitation or ion exchange are not so effective, especially in highly diluted solutions 7,8,11. Biosorption, either with living or dead biomass, is an attractive alternative for wastewater treatment 10,12. The use of such biological materials can reduce the consumption of toxic chemicals. Many microorganisms have been described to accumulate or immobilize metals. For instance, cells of Lysinibacillus sphaericus (L. sphaericus) JG-A12 have shown high binding capacities for precious metals, e.g., Pd(II), Pt(II), Au (III), and other toxic metals like Pb(II) or U(VI) 4,13, cells of Bacillus megaterium for Cr(VI) 14, cells of Saccharomyces cerevisiae for Pt(II) and Pd(II) 15, and Chlorella vulgar for Au(III) and U(VI) 16,17. The binding of previous metals like Au(III), Pd(II), and Pt(II) has also been reported for Desulfovibrio desulfuricans18 and for L. sphaericus JG-B53 19,20. Nevertheless, not all microbes bind high amounts of metals and their application as sorptive material is limited 12,21. Furthermore, metal binding capacity depends on different parameters, e.g., cell composition, the used bio-component, or environmental and experimental conditions (pH, ionic strength, temperature etc.). The study of isolated cell wall fragments 22,23, like membrane lipids, peptidoglycan, proteins, or other components, helps to understand the metal binding processes of complex constructed whole cells 8,21.
The cell components focused on in this study are S-layer proteins. S-layer proteins are parts of the outer cell envelope of many bacteria and archaea, and they constitute about 15 - 20% of the total protein mass of these organisms. As the first interface to the environment, these cell compounds strongly influence the bacterial sorption properties 3. S-layer proteins with molecular weights ranging from forty to hundreds of kDa are produced within the cell, but are assembled outside where they are able to form layers on the lipid membranes or polymeric cell wall components. Once isolated, nearly all S-layer proteins have the intrinsic property to spontaneously self-assemble in suspension, at interfaces, or on surfaces forming planar or tube-like structures 3. The thickness of the protein monolayer depends on the bacteria and is within a range of 5 - 25 nm 24. In general, the formed S-layer protein structures can have an oblique (p1 or p2), square (p4), or hexagonal (p3 or p6) symmetry with lattice constants of 2.5 to 35 nm 3,24. The lattice formation seems to be in many cases dependent on divalent cations and mainly on Ca2+ 25,26, Raff, J. et al. S-layer based nanocomposites for industrial applications in Protein-based Engineered Nanostructures. (eds Tijana Z. Grove &#38; Aitziber L. Cortajarena) (Springer, 2016 (submitted)). Nevertheless, the full reaction cascade of monomer folding, monomer-monomer interaction, the formation of a lattice, and the role of different metals, especially of divalent cations such as Ca2+ and Mg2+, are still not fully understood.
The gram-positive strain L. sphaericus JG-B53 (renamed from Bacillus sphaericus after new phylogenetic classification) 27 was isolated from the uranium mining waste pile &#34;Haberland&#34; (Johanngeorgenstadt, Saxony, Germany) 4,28,29. Its functional S-layer protein (Slp1) possesses a square lattice, a molecular weight of 116 kDa 30, and a thickness of &#8776; 10 nm on living bacteria cells 31. In previous studies, the in vitro formation of a closed and stable protein layer with a thickness of approximately 10 nm was achieved in less than 10 min&#160;19. The related strain L. sphaericus JG-A12, also an isolate from the &#34;Haberland&#34; pile, possesses high metal binding capacities and its isolated S-layer protein has shown a high chemical and mechanical stability and good sorption rates for precious metals like Au(III), Pt(II), and Pd(II) 4,32,33. This binding of precious metals is more or less specific for some metals and depends on the availability of functional groups on the outer and inner protein surface of the polymer and in its pores, ionic strength, and the pH value. Relevant functional groups for metal interaction by the proteins are COOH-, NH2-, OH-, PO4-, SO4-, and SO-. In principle, metal binding capacities open a wide spectrum of applications,Raff, J. et al. S-layer based nanocomposites for industrial applications in Protein-based Engineered Nanostructures. (eds Tijana Z. Grove &#38; Aitziber L. Cortajarena) (Springer, 2016 (submitted)).e.g., as biosorptive components for removal or recovery of dissolved toxic or valuable metals, templates for synthesis or defined deposition of regularly structured metallic nanoparticles (NPs) for catalysis, and other bio-engineered materials like bio-sensory layers 3,5,18,33. Regularly arranged NP arrays like Au(0)-NPs could be used for major applications ranging from molecular electronics and biosensors, ultrahigh density storage devices, and catalysts for CO-oxidation 34-37. The development of such applications and smart design of these materials necessitates a deeper understanding of the underlying metal binding mechanisms.
A prerequisite for the development of such bio-based materials is the reliable implementation of an interface layer between the biomolecule and the technical surface 38,39. For example, polyelectrolytes assembled with the layer-by-layer (LbL) technique 40,41 have been used as an interface layer for recrystallization of S-layer proteins 39. Such an interface offers a relatively easy way to perform the protein coating in a reproducible and quantitative way. By performing different experiments with and without modification with adhesive promoters, it is possible to make statements regarding coating kinetics, layer stability, and interaction of metals with biomolecules 19,42,Raff, J. et al. S-layer based nanocomposites for industrial applications in Protein-based Engineered Nanostructures. (eds Tijana Z. Grove &#38; Aitziber L. Cortajarena) (Springer, 2016 (submitted)). However, the complex mechanism of the protein adsorption and protein-surface interaction is not completely understood. Especially information on conformation, pattern orientation, and coating densities is still missing.
Quartz crystal microbalance with dissipation monitoring (QCM-D) technique has attracted attention in the recent years as a tool for studying protein adsorption, coating kinetics, and interaction processes on the nanometer scale 19,43-45. This technique allows for the detailed detection of mass adsorption in real-time, and can be used as an indicator for the protein self-assembling process and coupling of functional molecules on protein lattices 19,20,42,46-48. In addition, QCM-D measurements open the possibility to study metal interaction processes with the proteinaceous layer under natural biological conditions. In a recent study, the interaction of the S-layer protein with selected metals like Eu(III), Au(III), Pd(II),and Pt(II) has been studied with QCM-D 19,20. The adsorbed protein layer can serve as a simplified model of a cell wall of gram-positive bacteria. The study of this single component can contribute to a deeper understanding of metal interaction. However, solely QCM-D experiments do not allow statements regarding surface structures and influences of metals to protein. Other techniques are necessary to obtain such information. One possibility for imaging bio-nanostructures and obtaining information on structural properties is the atomic force microscopy (AFM).
The objective of the presented study was to investigate the sorption of gold (Au(III) and Au(0)-NPs) to S-layer proteins, in particular Slp1 of L. sphaericus JG-B53. Experiments were done with suspended proteins on batch scale in a pH range of 2.0 - 5.0 using ICP-MS and with immobilized S-layers using QCM-D. Additionally, the influence of metal salt solution on the lattice stability was investigated with subsequent AFM studies. The combination of these techniques contributes to a better understanding of in vitro metal interaction processes as a tool for learning more about binding events on whole bacterial cells regarding specific metal affinities. This knowledge is not only crucial for the development of applicable filter materials for the recovery of metals for environmental protection and the conservation of resources 49, but also for the development of arrays of highly ordered metallic NPs for various technical applications.

<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.

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-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 ...

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

11.3K Views.  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.

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

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.

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

Engineering

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.

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

Biochemistry

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.

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.

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.

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.

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 ...

Bioengineering

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.

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.

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.

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

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

Summary

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Chapters in this video

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

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

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

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

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

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

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

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