Name: water in oil emulsions: a new system for assembling water-soluble chlorophyll-binding proteins with hydrophobic pigments
Uploaded: 2016-03-21T14:00:00
Description: Watch this Scientific Journal Video about Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments Video at JoVE.com

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.<...

<ol>
	<li>Bryant, D. A., Frigaard, N. -. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokaryotic+photosynthesis+and+phototrophy+illuminated.">Prokaryotic photosynthesis and phototrophy illuminated.</a> Trends Microbiol. 14, 488-496 (2006).</li><li>Tronrud, D. E., Wen, J. Z., Gay, L., Blankenship, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+basis+for+the+difference+in+absorbance+spectra+for+the+FMO+antenna+protein+from+various+green+sulfur+bacteria.">The structural basis for the difference in absorbance spectra for the FMO antenna protein from various green sulfur bacteria.</a> Photosynth. 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. Plant Physiol. 168, 1462-1472 (2011).</li><li>Satoh, H., Uchida, A., Nakayama, K., Okada, M. <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+protein+in+Brassicaceae+plants+is+a+stress-induced+chlorophyll-binding+protein.">Water-soluble chlorophyll protein in Brassicaceae plants is a stress-induced chlorophyll-binding protein.</a> Plant Cell Physiol. 42, 906-911 (2001).</li><li>Bektas, I., Fellenberg, C., Paulsen, H. <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+protein+(WSCP)+of+Arabidopsis+is+expressed+in+the+gynoecium+and+developing+silique.">Water-soluble chlorophyll protein (WSCP) of Arabidopsis is expressed in the gynoecium and developing silique.</a> Planta. 236, 251-259 (2012).</li><li>Damaraju, S., Schlede, S., Eckhardt, U., Lokstein, H., Grimm, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+the+water+soluble+chlorophyll-binding+protein+in+plants.">Functions of the water soluble chlorophyll-binding protein in plants.</a> J. Plant Physiol. 168, 1444-1451 (2011).</li><li>Horigome, D., 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=Structural+mechanism+and+photoprotective+function+of+water-soluble+chlorophyll-binding+protein.">Structural mechanism and photoprotective function of water-soluble chlorophyll-binding protein.</a> J. Biol. Chem. 282, 6525-6531 (2007).</li><li>Reinbothe, C., Satoh, H., Alcaraz, J. -. P., Reinbothe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Role+of+Water-Soluble+Chlorophyll+Proteins+in+the+Transitory+Storage+of+Chorophyllide.">A Novel Role of Water-Soluble Chlorophyll Proteins in the Transitory Storage of Chorophyllide.</a> Plant Physiol. 134, 1355-1365 (2004).</li><li>Satoh, H., Nakayama, K., Okada, M. <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+protein,+a+putative+carrier+of+chlorophyll+molecules+in+cauliflower.">Molecular cloning and functional expression of a water-soluble chlorophyll protein, a putative carrier of chlorophyll molecules in cauliflower.</a> J. Biol. Chem. 273, 30568-30575 (1998).</li><li>Schmidt, K., Fufezan, C., Krieger-Liszkay, A., Satoh, H., Paulsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+water-soluble+chlorophyll+protein+from+Brassica+oleracea+var.+Botrys+binds+various+chlorophyll+derivatives.">Recombinant water-soluble chlorophyll protein from Brassica oleracea var. 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. Plant Physiol. 170, 406-412 (2013).</li><li>Boex-Fontvieille, E., Rustgi, S., von Wettstein, D., Reinbothe, S., Reinbothe, C. <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+protein+is+involved+in+herbivore+resistance+activation+during+greening+of+Arabidopsis+thaliana.">Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 7303-7308 (2015).</li><li>Hughes, J. L., 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=Magneto-optic+spectroscopy+of+a+protein+tetramer+binding+two+exciton-coupled+chlorophylls.">Magneto-optic spectroscopy of a protein tetramer binding two exciton-coupled chlorophylls.</a> J. Am. Chem. 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. Chem. 267, 22043-22047 (1992).</li><li>Kamimura, Y., Mori, T., Yamasaki, T., Katoh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+properties+and+a+possible+function+of+a+water-soluble+chlorophyll+a/b-protein+from+brussels+sprouts.">Isolation, properties and a possible function of a water-soluble chlorophyll a/b-protein from brussels sprouts.</a> Plant Cell Physiol. 38, 133-138 (1997).</li><li>Murata, T., Itoh, R., Yakushiji, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+water-soluble+chlorophyll-proteins+from+Lepidium+virginicum.">Crystallization of water-soluble chlorophyll-proteins from Lepidium virginicum.</a> Biochim. Biophys. Acta. 593, 167-170 (1980).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="614">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:119px;">M5904</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Span80</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:119px;">85548</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween80</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:119px;">P8074</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bio-Scale Mini Profinity eXact Cartridges</td>
			<td style="width:83px;">Bio Rad</td>
			<td style="width:119px;">10011164</td>
			<td style="width:196px;">Affinity chromatography for WSCP purification with native sequence.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">His Trap HF column</td>
			<td style="width:83px;">GE Healthcare Life Science</td>
			<td style="width:119px;">17-5248-02</td>
			<td style="width:196px;">Affinity chromatography for WSCP purification with His-tag</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">DEAE Sepharose Fast Flow</td>
			<td style="width:83px;">GE Healthcare Life Science</td>
			<td style="width:119px;">17-0709-01</td>
			<td style="width:196px;">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...

Watch this Scientific Journal Video about Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments Video at JoVE.com

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments Video (Video) | JoVE

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.

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. ...

Research

Education

JoVE Core

JoVE Journal

Biology

Chemistry

Cellular Processes

Photosynthesis

SCIENTISTS IN ACTION

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems Video (Video) | JoVE

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS Video (Video) | JoVE

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry Video (Video) | JoVE

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

Synthesis of a Water-soluble Metalâ€“Organic Complex Array Video (Video) | JoVE

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives Video (Video) | JoVE

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous ...

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy Video (Video) | JoVE

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

Heterogeneous Removal of Water-Soluble Ruthenium Olefin Metathesis Catalyst from Aqueous Media Via Host-Guest Interaction

Heterogeneous Removal of Water-Soluble Ruthenium Olefin Metathesis Catalyst from Aqueous Media Via Host-Guest Interaction Video (Video) | JoVE

A highly efficient transition metal catalyst removal method is developed. The water-soluble catalyst contains a newly-designed NHC ligand for the catalyst ...

Stereolithographic 3D Printing with Renewable Acrylates

Stereolithographic 3D Printing with Renewable Acrylates Video (Video) | JoVE

The accessibility of cost-competitive renewable materials and their application in additive manufacturing is essential for an efficient biobased economy. ...

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

A New Straightforward Method for Lipophilicity LogP Measurement using 19F NMR Spectroscopy Video (Video) | JoVE

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates Video (Video) | JoVE

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...