This work was supported by the Marie Curie International Reintegration Grant and by the German-Israel Foundation. We acknowledge Mr. Yair Razvag for AFM analysis.

1. Self-assembly of Homoaromatic Dipeptides
<ol>
	<li>Weigh the desired peptide in its lyophilized form (e.g. NH2-Phe-Phe-OH, Boc-Phe-Phe-COOH) and prepare a stock solution by dissolving the peptide in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) to the appropriate concentration (e.g. 100 mg/ml for NH2-Phe-Phe-OH and Boc-Phe-Phe-COOH)7,17,46.</li>
	<li>Mix the solution&#160;using vortex and place on the bench until the peptide&#160;is&#160;completely dissolved and the solut...

In summary, this paper demonstrates the ease in which peptide-based assemblies can be formed in vitro. The process involves commercially available peptides and solvents, and it occurs spontaneously under ambient conditions, upon the addition of a polar solvent to the test tube. It is crucial to use HFP as a solvent of the peptides, due to the low solubility of the peptides in other organic solvents. In addition, due to the high volatility of HFP it is necessary to prepare fresh stock solution for each experiment...

Nature forms ordered and functional structures by the process of biomolecular self-assembly. Understanding the forces that govern this spontaneous process may lead to the ability to mimic self-assembly in vitro and consequently to major advances in the area of material sciences10,11. Peptides, specifically, hold great promise as a biomolecular building block, since they present large structural diversity, ease of chemical synthesis, and can easily be functionalized with biological and chemical entities. The field of peptide self-assembly was pioneered by Ghadiri and his colleagues, who demonstrated the self-assembly of peptide nanotubes by cyclic peptides with alternating D- and L-amino acids12. Other successful approaches to the design of peptide assemblies include linear bolaamphiphile peptides5,&#160;amphiphiles (AP)6, nonconjugated self-complementary ionic peptides13, surfactant-like peptides4,14, and diblock copolypeptides15.
A more recent approach involves the self-assembly of short aromatic peptides, termed homoaromatic dipeptides. These peptides comprise only two amino acids with aromatic nature (e.g. Phe-Phe, tert-butyl dicarbonate (Boc)-Phe-Phe)7,8,16-21. The structures formed by these homoaromatic peptides include tubular structures, spheres, sheet-like assemblies and fibers6,8,15,21-32. The fibers in some cases generate a fibril mesh that yields a hydrogel33-37. These assemblies have been exploited for applications of biosensing, drug delivery, molecular electronics, etc.38-45
This paper describes the experimental steps needed in order to start the spontaneous self-assembly of homoaromatic peptides. In addition, it presents the process of peptide coassembly. This process involves the self-assembly of more than one type of peptide monomer.
Our demonstration includes the coassembly of two commercially available peptides: the diphenylalanine peptide (NH2-Phe-Phe-COOH) and its Boc protected analogue (Boc-Phe-Phe-OH). Each of the peptides self-assembles into a supermolecular structure: the diphenylalanine peptide forms tubular assemblies and the Boc-Phe-Phe-OH peptide self-assembles into either spheres or fibers depending on the solvent7,17,46. We blended the two peptides in certain ratios and characterized the resulted assemblies by electron microscopy, force microscopy and FT-IR spectroscopy. The methods demonstrated the formation of a peptide-based structure which is comprised of spherical elements with a diameter of several microns (1-4 &#956;m) that are connected by elongated assemblies with a diameter of a few hundred nanometers (~300-800 nm). The assemblies resemble beaded strings in their morphology,&#160;as the spherical structures seem to be threaded on the elongated assemblies. We therefore termed these assemblies &#34;biomolecular necklaces&#34;. The &#34;biomolecular necklaces&#34; might serve as a new biomaterial, as a drug delivery agent or as a scaffold for electronic applications. Moreover, the procedure that leads to the self-assembly of peptides may be utilized with other classes of peptides and biomolecules. It may lead to a better understanding of the forces involved in self-assembly and the formation of new ordered structures.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NH2-Phe-Phe-OH</td>
			<td>Bachem</td>
			<td>G-2925.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Boc-Phe-Phe-OH</td>
			<td>Bachem</td>
			<td>A-3205.0005</td>
			<td></td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>52512-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute (Dehydrated) AR sterile</td>
			<td>Bio-Lab Ltd.</td>
			<td>52555</td>
			<td>Blending with TDW for the preparation of 50% solution</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>73943</td>
			<td>For negative staining. It is possible to work without it.</td>
		</tr>
		<tr>
			<td>glass cover slip</td>
			<td>Marienfeld Laboratory Glassware</td>
			<td>110590</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>FCF200-Cu-50</td>
			<td>Formvar/Carbon 200 Mesh, Cu</td>
		</tr>
		<tr>
			<td>Quantitive filter paper</td>
			<td>Whatman</td>
			<td>1001055</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium Oxide (D2O)</td>
			<td>Sigma-Aldrich</td>
			<td>151882-100G</td>
			<td>99.9 atom % D</td>
		</tr>
		<tr>
			<td>CaF2 window</td>
			<td>PIKE Technologies</td>
			<td>160-1212</td>
			<td>25 mm x 2 mm window. For FT-IR measurments</td>
		</tr>
		<tr>
			<td>AFM tips</td>
			<td>NanoScience Instruments</td>
			<td>CFMR</td>
			<td>Aspire probes, CFMR-25 series</td>
		</tr>
		<tr>
			<td>Filter units</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td>Millex-GV, 0.22 &#956;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>FEI</td>
			<td></td>
			<td>Quanta 200 ESEM</td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>FEI</td>
			<td></td>
			<td>Tecnai T12 G2 Spirit</td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>JPK Instruments</td>
			<td></td>
			<td>A JPK NanoWizard3</td>
		</tr>
		<tr>
			<td>FT-IR</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Nicolet 6700 advanced gold spectrometer</td>
		</tr>
		<tr>
			<td>FT-IR Purge</td>
			<td>Parker</td>
			<td></td>
			<td>BALSTON FT-IR Purge Gas Generator model 75-52</td>
		</tr>
		<tr>
			<td>OMNIC (Nicolet) software</td>
			<td>Thermo Nicolet Corporation</td>
			<td></td>
			<td>For FT-IR spectra analysis</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Wisd Laboratory Equipment</td>
			<td></td>
			<td>ViseMix VM</td>
		</tr>
		<tr>
			<td>Weight</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>NewClassic MS</td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Polaron</td>
			<td></td>
			<td>SC7640 Sputter Coater</td>
		</tr>
	</tbody>
</table>

formation of ordered biomolecular structures by the self-assembly of short peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes a method for the formation of ordered structures at the nano-and micrometer scale by the self-assembly of peptides. In order to demonstrate this simple process we present and characterize the coassembly of two simple aromatic peptides (Figure 1). One of the peptides is the NH2-Phe-Phe-OH (diphenylalanine) peptide, which can self-assemble in an aqueous solution into hollow tubular structures with nanometric dimensions7. The other peptide is its Boc protected anal...

Watch this Scientific Journal Video about Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides at JoVE.com

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

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12.7K ビュー.  The Hebrew University of Jerusalem. 本論文では、自己組織化の自発的プロセスによる高度に秩序ペプチドベースの構造の形成を説明しています。この方法は、市販のペプチドおよび共通実験装置を利用しています。この技術は、ペプチドの多種多様に適用することができる、新たなペプチドベースアセンブリの発見につながることができる。 

ビデオ: Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

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 ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the &#34;A-form&#34; conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living ...