DN acknowledges support from EU FP7 projects PEPDIODE (GA 256672) and REGPOT-2012-2013-1(GA 316157), and a personal research grant (No. 268/10) from the Israel Science Foundation. We thank Prof. Shmuel Rubinstein, School of Engineering and Applied Sciences, Harvard University, Cambridge MA, USA for taking the confocal microscopy images.

1. Preparing Chl a Stock Solutions
<ol>
	<li>CRITICAL STEP: Perform all the steps of chlorophyll preparation in a chemical hood, under green light (520 nm) or in the dark in order to minimize photodamage. Always add Nitrogen or Argon before freezing the pigments for storage. Ensure that all solvents are analytical grade.</li>
	<li>Weigh about 5 mg of lyophilized Spirulina platensis cells or other cyanobacterium cells containing only Chl a in thylakoid membranes and crush it using a mortar and pestle.</li>
	<li>Load crushed cells onto a glass column and wash with about 50-100 ml of 100% acetone in order to remove carotenoids. Discard the eluted orange/green fraction. 
	Note: If the orange fraction is not eluted with 100 ml of acetone continue washing the cells with acetone until green fraction starts to elute.</li>
	<li>Exchange acetone with 100% methanol and collect the green fraction containing Chl a. The volume of eluted fraction may vary between 50-100 ml. At the beginning, the eluted fraction has a dark green color, which changes into pale green at the end of elution. When the color of eluted fraction turns into pale green stop the elution.</li>
	<li>Evaporate the methanol using a rotary evaporator until the extract is completely dry. Do not apply heat to the solution; ensure that the evaporator&#39;s water bath temperature does not exceed 30 &#176;C. 
	Note: The time of evaporation depends on the volume of methanol fraction being evaporated and may vary between 10-60 min. It is important to dry the extract completely.</li>
	<li>Dissolve the pigments from the dried extract in a small volume of diethyl ether (about 5-10 ml), and filter through cotton wool. Ensure that pigments are completely dissolved in ether before filtering.</li>
	<li>Evaporate the diethyl ether until the pigments are completely dry (10-30 min). 
	Note: The dry pigments can be purged with and kept under Nitrogen or Argon at -20 &#176;C, in the dark until further processing.</li>
	<li>Dissolve the dry pigments in the smallest volume possible of 100% methanol (about 1 ml), even if not everything is completely suspended. Add 4 ml of acetone to the solution, and flick the glass gently in order to completely dissolve the pigments.</li>
	<li>Using a Pasteur pipette, load the sample gently onto a column of DEAE sepharose equilibrated in 100% acetone.</li>
	<li>Elute carotenoids (an orange-yellow band) with 100% acetone. Then, elute Chl a (green band) with 3:1 v/v acetone/methanol mixture. 
	Note: The volume of acetone and acetone/methanol mixture is approximately equivalent to the volume of DEAE sepharose loaded on the column.</li>
	<li>Verify Chl a purity by thin layer chromatography using a 68:25:5:2 dichloromethane/n-hexane /isopropanol /methanol (v/v) mixture as eluent 17.</li>
	<li>Evaporate the solvent using a rotary evaporator until the Chl a is completely dry (10-60 min). 
	Note: The dry Chl a can be purged with nitrogen or argon and stored under argon atmosphere at -20 &#176;C in the dark.</li>
	<li>Prepare Chl a stock solution by re-dissolving the dry Chl a in 2-4 ml of 100% ethanol. 
	Note: The extinction coefficient of Chl a at 663 nm is 74,400 cm-1M-1 (83.3 cm-1 (mg/ml)-1) in ethanol. A typical stock solution should have an OD of 1,860 corresponding to a concentration of 25 mM (23 mg/ml). Adding 20 &#181;l of this stock to an emulsion containing 5 ml of organic phase, and 1 mg of WSCP in 1 ml of aqueous phase results in a mixture with 10 fold molar excess of Chl a vs. WSCP.</li>
</ol>
2. Preparing Organic Phase of the Emulsion
Note: The organic phase of the emulsion is composed of mineral oil containing 4.5% (v/v) Span 80, and 0.4% (v/v) Tween 80.
<ol>
	<li>Weigh in a 50 ml tube 0.2 g of Tween 80, 1.8 g of Span 80, and 38 g of mineral oil. Mix well all components and cool down on ice. 
	Note: The organic phase can be stored in 4 &#176;C up to one week.</li>
</ol>
3. Preparing the Aqueous Phase of the Emulsion
Note: The aqueous phase of the emulsion may be composed of either purified WSCP, or crude extract of bacteria overexpressing WSCP.
<ol>
	<li>Preparing an aqueous phase containing purified WSCP.
	<ol>
		<li>Grow E.coli BL21 bacteria containing WSCP plasmid 12 in 1 L of LB medium at 37 &#176;C until OD of 0.3-0.6.</li>
		<li>Induce protein expression by adding 1 mM IPTG. After induction grow bacteria at 30 &#176;C for 12-16 hr.</li>
		<li>Harvest bacteria by centrifugation at 5,000 x g for 10 min at 4 &#176;C.</li>
		<li>Dissolve the pellet in binding buffer and sonicate on ice (30 sec on, 15 sec off, five times). Use 10 ml of buffer for the pellet obtained from centrifugation of 250 ml of LB medium with cells overexpressing the protein. 
		Note: Depending on purification method, the binding buffer may be composed of 100 mM phosphate buffer, pH 7.2 or 50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM EDTA.</li>
		<li>Spin down the cell lysate at 12,000 x g for 30 min at 4 &#176;C.</li>
		<li>Purify WSCP by affinity chromatography. Depending on the tag fused to WSCP, purify recombinant proteins using the appropriate commercially available affinity chromatography system for protein purification following manufacturer instructions.</li>
		<li>For emulsion preparation, use purified WSCP in 50 mM phosphate buffer, pH 7.8. Ensure that the final protein amount used for reconstitution is 0.5-1.0 mg per 1 ml of buffer.</li>
	</ol>
	</li>
	<li>Preparing an aqueous phase containing crude bacterial lysate with WSCP.
	<ol>
		<li>Grow E.coli BL21 cells containing plasmid-expressing WSCP in 250 ml of LB medium at 37 &#176;C until OD 0.3-0.6.</li>
		<li>Induce protein expression with 1 mM IPTG and grow bacteria overnight at 30 &#176;C.</li>
		<li>Harvest bacterial cells by centrifugation at 5,000 x g for 10 min at 4 &#176;C.</li>
		<li>Dissolve the pellet in 1-2 ml of 50 mM sodium phosphate buffer pH 7.8, sonicate (30 sec on, 15 sec off, three times) and centrifuge at 12,000 x g for 30 min at 4 &#176;C.</li>
		<li>Prepare the aqueous phase of the emulsion by mixing 0.125 ml of supernatant with 0.875 ml of 50 mM sodium phosphate buffer pH 7.8.</li>
	</ol>
	</li>
</ol>
4. Assembly of WSCP with Chl a in Emulsion
<ol>
	<li>Transfer 5 ml of oil-surfactant mixture into a glass vial and cool it on ice. Before pipetting, verify that all the components of the organic phase are mixed thoroughly and there is no phase separation between surfactants and mineral oil.</li>
	<li>Add 1 ml of ice-cold aqueous phase prepared as in section 3 to 5 ml of organic phase.</li>
	<li>Mix both phases using a tissue homogenizer for 2 min at 9,500 rpm on ice.</li>
	<li>CRITICAL STEP: From this stage on, perform all further steps under green light (520 nm) in order to minimize photodamage. Add 20 &#181;l of 25 mM Chl a stock solution (see section 1.13) to the emulsion. Disperse by flicking and inverting the glass vial. Make sure that the Chl is evenly distributed in the emulsion.</li>
	<li>Incubate the emulsion for 1-2 hr on ice in the dark.</li>
	<li>In order to break down the emulsion and separate water droplets from the organic phase transfer the emulsion to 1.5 ml plastic tubes and centrifuge at 14,000 x g for 5 min at room temperature. 
	Note: If the assembly is successful, the lower aqueous phase should have a green color.</li>
	<li>Dispose the upper oil phase and add 1 ml of mineral oil. Mix well the mineral oil with the pelleted emulsion by vortex or by flipping the tube thoroughly. Spin down the sample at 14,000 x g for 5 min at room temperature. Repeat this step until a clear meniscus separating the aqueous and mineral oil phases, without any intermediate emulsion is obtained.</li>
	<li>Perform this step in a chemical hood. After the aqueous and mineral oil phases are clearly separated, remove mineral oil and add 1 ml of water-saturated diethyl ether. Vortex and spin down the sample at 14,000 x g for 5 min at room temperature. Repeat this step twice.</li>
	<li>After the second centrifugation, remove the diethyl ether and leave the tubes open for 5-20 min in the hood.</li>
	<li>Finally, load the aqueous phase containing the WSCP/Chl a complex onto a desalting column and elute with buffer appropriate for further experiments. 
	Note: The protein is stable in phosphate- and Tris buffers in a broad range of pH (6.0-7.5). The sample can be stored at 4 &#176;C protected from light up to one month.</li>
</ol>

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Res. 100, 79-87 (2009).</li><li>Schulte, T., Johanning, S., Hofmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+native+and+refolded+peridinin-chlorophyll-proteins+from+dinoflagellates.">Structure and function of native and refolded peridinin-chlorophyll-proteins from dinoflagellates.</a> Eur. J. Cell Biol. 89, 990-997 (2010).</li><li>Renger, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+soluble+chlorophyll+binding+protein+of+higher+plants:+a+most+suitable+model+system+for+basic+analyses+of+pigment-pigment+and+pigment-protein+interactions+in+chlorophyll+protein+complexes.">Water soluble chlorophyll binding protein of higher plants: a most suitable model system for basic analyses of pigment-pigment and pigment-protein interactions in chlorophyll protein complexes.</a> J. 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Botrys binds various chlorophyll derivatives.</a> Biochemistry. 42, 7427-7433 (2003).</li><li>Takahashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+characterization+and+analysis+of+the+intracellular+localization+of+a+water-soluble+Chl-binding+protein+from+Brussels+sprouts+(Brassica+oleracea+var.+gemmifera).">Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera).</a> Plant Cell Physiol. 53, 879-891 (2012).</li><li>Takahashi, S., Ono, M., Uchida, A., Nakayama, K., Satoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+functional+expression+of+a+water-soluble+chlorophyll-binding+protein+from+Japanese+wild+radish.">Molecular cloning and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild radish.</a> J. 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Soc. 128, 3649-3658 (2006).</li><li>Bednarczyk, D., Takahashi, S., Satoh, H., Noy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+water-soluble+chlorophyll-binding+proteins+with+native+hydrophobic+chlorophylls+in+water-in-oil+emulsions.">Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions.</a> BBA - Bioenergetics. 1847, 307-313 (2015).</li><li>Fiedor, L., Rosenbach-Belkin, V., Scherz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stereospecific+interaction+between+chlorophylls+and+chlorophyllase.+Possible+implication+for+chlorophyll+biosynthesis+and+degradation.">The stereospecific interaction between chlorophylls and chlorophyllase. Possible implication for chlorophyll biosynthesis and degradation.</a> J. Biol. 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The authors declare that they have no competing financial interests.

Our goal was to develop a new general system for assembly of water-soluble chlorophyll-binding proteins with hydrophobic pigments. Here it is shown that the new reconstitution system based on W/O emulsion is a general approach proven to work for assembly of WSCP apoproteins from Brussels sprouts, cauliflower, Japanese horseradish and Virginia pepperweed recombinantly expressed in E. coli. Here results are presented from reconstitution of 1 mg of WSCP with 10-fold molar excess of Chl a. However it is als...

Hydrophobic pigments such as chlorophylls (Chls), bacteriochlorophylls (BChls) and carotenoids are the primary cofactors in photosynthetic reaction centers and light harvesting proteins that carry out electron transport, and light energy capture and transfer. The reaction centers and most of the Chl-binding light harvesting complexes are transmembrane proteins. The Fenna-Matthews-Olson (FMO) protein of non-oxygenic photosynthetic green-sulfur bacteria 1,2, and the peridinin-Chl protein (PCP) of dinoflagellates 3 are exceptional examples of water soluble light harvesting proteins. The water-soluble chlorophyll binding proteins (WSCPs) of Brassicaceae, Polygonaceae, Chenopodiaceae and Amaranthaceae plants 4,5 are another unique example, yet in contrast to FMO and PCP, these are neither involved in light harvesting nor in any of the primary photosynthetic reaction, and their precise physiological functions are yet unclear 5-8. Their high Chl-binding affinity have led to a suggested function as transient carriers of Chls and Chl derivatives 9,10. Alternatively, it was hypothesized that WSCP plays a role in scavenging Chls in damaged cells and protects against Chl-induced photooxidative damage 7,11-13. More recently, it was suggested that WSCP functions as a protease inhibitor and plays a role during herbivore resistance as well regulates cell death during flower development 14. WSCPs are divided into two main classes according to their photophysical properties. The first class (class I, e.g. from Chenopodium album) may undergo photoconversion upon illumination. Class II WSCPs from Brassica plants, that do not undergo photoconversion 5,10, are further subdivided into class IIa (e.g., from Brassica oleracea, Raphanus sativus) and IIb (e.g., from Lepidium virginicum). The structure of class IIb WSCP from Lepidium virginicum was solved by X-ray crystallography at 2.0 &#197; resolution 8. It reveals a symmetric homotetramer in which the protein subunits form a hydrophobic core. Each subunit binds a single Chl which results in a tight arrangement of four closely packed Chls within the core.This simple all Chl arrangement makes WSCPs a potentially useful model system for studying binding and assembly of Chl-protein complexes, and the effects of neighboring Chls and protein environments on the spectral and electronic properties of individual Chls. Furthermore, it may provide templates for constructing artificial Chl-binding proteins that may be used for light-harvesting modules in artificial photosynthetic devices.
Rigorous studies of native WSCPs are not feasible because the complexes purified from plants always contain a heterogeneous mixture of tetramers with different combinations of Chl a and Chl b 9. Thus, a method for assembling recombinantly expressed WSCPs with Chls in vitro is required. This is challenged by the negligible water-solubility of Chls which makes it impossible to assemble the complex in&#160;vitro by simply mixing the water-soluble apoproteins with pigments in aqueous solutions. In&#160;vitro assembly by mixing the apoproteins with thylakoid membranes 15 was demonstrated, but this method is limited to the native Chls present in the thylakoids. Schmidt et al. reported on assembling several Chl and BChl derivatives with WSCP from cauliflower (CaWSCP) by recombinantly expressing a histidine-tagged protein in E. coli immobilizing it onto a Ni-affinity column and introducing Chl derivatives solubilized in detergents 11. Successfully reconstitution of recombinant WSCPs from A. thaliana 6, and Brussels sprouts (BoWSCP), Japanese wild radish (RshWSCP) and Virginia pepperweed (LvWSCP) by a similar method were also reported.
Here, we present a novel, general, straightforward method for assembling Chls with WSCP that does not require tagging or immobilizing the proteins. It relies on preparing emulsions from their aqueous solutions of the water-soluble apoproteins in mineral oil. The proteins are thus encapsulated in water-in-oil (W/O) microdroplets with very high surface to volume ratio 16. The hydrophobic cofactors are then dissolved in the oil and are readily introduced into the droplets from the oil phase. We report on using the method for assembling of several variants of WSCP apoproteins recombinantly expressed in E. coli with Chl a. We demonstrate the assembly from crude lysate of WSCP-overexpressing bacteria which may be used as a screening system for developing novel Chl binding proteins.

<table><tbody><tr><td>Mineral oil</td><td>Sigma</td><td>M5904</td><td></td></tr><tr><td>Span80</td><td>Sigma</td><td>85548</td><td></td></tr><tr><td>Tween80</td><td>Sigma</td><td>P8074</td><td></td></tr><tr><td>Bio-Scale Mini Profinity eXact Cartridges</td><td>Bio Rad</td><td>10011164</td><td>Affinity chromatography for WSCP purification with native sequence.</td></tr><tr><td>His Trap HF column</td><td>GE Healthcare Life Science</td><td>17-5248-02</td><td>Affinity chromatography for WSCP purification with His-tag</td></tr><tr><td>DEAE Sepharose Fast Flow</td><td>GE Healthcare Life Science</td><td>17-0709-01</td><td>Chromatography medium for chlorophyll purification</td></tr></tbody></table>

water in oil emulsions: a new system for assembling water-soluble chlorophyll-binding proteins with hydrophobic pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

Recombinant WSCP apoproteins were assembled with Chl a in W/O emulsions according to the protocol described in the previous section. The protocol was implemented using aqueous phases containing either pure WSCPs, or lysates E.coli cells overexpressing WSCP (Figure 1). The protocol is simple, fast and does not require any special equipment except a tissue homogenizer.
The absorbance and CD spect...

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Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

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14.6K Views.  Weizmann Institute of Science.  Dit manuscript beschrijft een eenvoudige en high-throughput werkwijze voor het samenstellen in water oplosbare eiwitten met hydrofobe pigmenten die is gebaseerd op water-in-olie-emulsies. We tonen de effectiviteit van de werkwijze voor de assemblage van natieve chlorofyl vier varianten van recombinant water oplosbare chlorofyl-bindende eiwitten (WSCPs) van Brassica planten tot expressie gebracht in E. coli. 

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A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

Biochemistry

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Neuroscience

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds &#8211; PP2, rottlerin, and curcumin &#8211; in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.

This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings.

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic ...

Medicine

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

Bioengineering

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

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