Name: separation of spinach thylakoid protein complexes by native green gel electrophoresis and band characterization using time-correlated single photon counting
Uploaded: 2019-02-14T14:00:00
Description: Watch this Scientific Journal Video about Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting a

Funding and support were provided by the Department of Chemistry at Michigan State University.

1. Stock Solutions Preparation for Pouring Native Green Gels
<ol>
	<li>Prepare a 4x concentrated buffer solution for resolving gels consisting of 40% glycerol, 200 mM glycine, and 100 mM Tris buffered to pH 8.3.</li>
	<li>Prepare a 4x concentrated buffer solution for stacking gels consisting of 40% glycerol, 200 mM glycine, and 100 mM Tris buffered to pH 6.3.</li>
	<li>Store these buffers at 4 &#176;C to prevent the growth of mold. 
	Note: The buffers are stable for months at 4 &#176;C, so preparation of 100-200 m...

<ol>
	<li>Yamori, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthetic+response+to+fluctuating+environments+and+photoprotective+strategies+under+abiotic+stress.">Photosynthetic response to fluctuating environments and photoprotective strategies under abiotic stress.</a> Journal of Plant Research. 129 (3), 379-395 (2016).</li><li>Kim, J. H., Nemson, J. A., Melis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosystem+II+reaction+center+damage+and+repair+in+Dunaliella+salina+(Green+Alga)+(analysis+under+physiological+and+irradiance-stress+conditions).">Photosystem II reaction center damage and repair in Dunaliella salina (Green Alga) (analysis under physiological and irradiance-stress conditions).</a> Plant Physiology. 103 (1), 181-189 (1993).</li><li>Sundby, C., McCaffery, S., Anderson, J. 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L., Br&#228;utigam, K., Pfannschmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthetic+acclimation:+state+transitions+and+adjustment+of+photosystem+stoichiometry-functional+relationships+between+short-term+and+longterm+light+quality+acclimation+in+plants.">Photosynthetic acclimation: state transitions and adjustment of photosystem stoichiometry-functional relationships between short-term and longterm light quality acclimation in plants.</a> FEBS Journal. 275 (6), 1080-1088 (2008).</li><li>Horton, P., Ruban, A. V., Walters, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+light+harvesting+in+green+plants.">Regulation of light harvesting in green plants.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 47, 655-684 (1996).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annual Review of Plant Biology. 59, 89-113 (2008).</li><li>Williams, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+and+interaction+of+the+two+photosystems+in+photosynthesis.">Spatial organization and interaction of the two photosystems in photosynthesis.</a> Nature. 225 (5239), 1214-1217 (1970).</li><li>Bellafiore, S., Barneche, F., Peltier, G., Rochaix, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+transitions+and+light+adaptation+require+chloroplast+thylakoid+protein+kinase+STN7.">State transitions and light adaptation require chloroplast thylakoid protein kinase STN7.</a> Nature. 433 (7028), 892-895 (2005).</li><li>Shapiguzov, A., 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=The+PPH1+phosphatase+is+specifically+involved+in+LHCII+dephosphorylation+and+state+transitions+in+Arabidopsis.">The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis.</a> Proceedings of the National Academy of Sciences USA. 107 (10), 4782-4787 (2010).</li><li>Pribil, M., Pesaresi, P., Hertle, A., Barbato, R., Leister, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+plastid+protein+phosphatase+TAP38+in+LHCII+dephosphorylation+and+thylakoid+electron+flow.">Role of plastid protein phosphatase TAP38 in LHCII dephosphorylation and thylakoid electron flow.</a> Public Library of Science Biology. 8 (1), (2010).</li><li>Larsson, U. K., Jergil, B., Andersson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+lateral+distribution+of+the+light-harvesting+chlorophyll-a/b-protein+complex+induced+by+its+phosphorylation.">Changes in the lateral distribution of the light-harvesting chlorophyll-a/b-protein complex induced by its phosphorylation.</a> European Journal of Biochemistry. 136 (1), 25-29 (1983).</li><li>Minagawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+transitions--the+molecular+remodeling+of+photosynthetic+supercomplexes+that+controls+energy+flow+in+the+chloroplast.">State transitions--the molecular remodeling of photosynthetic supercomplexes that controls energy flow in the chloroplast.</a> Biochimica et Biophysica Acta. 1807 (8), 897-905 (2011).</li><li>Ruban, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonphotochemical+Chlorophyll+Fluorescence+Quenching:+Mechanism+and+Effectiveness+in+Protecting+Plants+from+Photodamage.">Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage.</a> Plant Physiology. 170 (4), 1903-1916 (2016).</li><li>Rokka, A., Suorsa, M., Saleem, A., Battchikova, N., Aro, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+assembly+of+thylakoid+protein+complexes:+multiple+assembly+steps+of+photosystem+II.">Synthesis and assembly of thylakoid protein complexes: multiple assembly steps of photosystem II.</a> Biochemical Journal. 388 (Pt 1), 159-168 (2005).</li><li>Li, L., Aro, E. M., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Photodamage+and+Protein+Turnover+in+Photoinhibition.">Mechanisms of Photodamage and Protein Turnover in Photoinhibition.</a> Trends in Plant Science. , (2018).</li><li>Dunahay, T. G., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+photosystem+I+complexes+from+octyl+glucoside/sodium+dodecyl+sulfate+solubilized+spinach+thylakoids:+characterization+and+reconstitution+into+liposomes.">Isolation of photosystem I complexes from octyl glucoside/sodium dodecyl sulfate solubilized spinach thylakoids: characterization and reconstitution into liposomes.</a> Plant Physiology. 78 (3), 606-613 (1985).</li><li>Allen, K. D., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+16+to+20+chlorophyllprotein+complexes+using+a+low+ionic+strength+native+green+gel+system.">Resolution of 16 to 20 chlorophyllprotein complexes using a low ionic strength native green gel system.</a> Analytical Biochemistry. 194 (1), 214-222 (1991).</li><li>Peter, G. F., Thornber, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+composition+and+organization+of+higher+plant+photosystem+II+light-harvesting+pigment-proteins.">Biochemical composition and organization of higher plant photosystem II light-harvesting pigment-proteins.</a> Journal of Biological Chemistry. 266 (25), 16745-16754 (1991).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Analytical Biochemistry. 199, 223-231 (1991).</li><li>Schwarz, E. M., Tietz, S., Froehlich, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosystem+I-LHCII+megacomplexes+respond+to+high+light+and+aging+in+plants.">Photosystem I-LHCII megacomplexes respond to high light and aging in plants.</a> Photosynthesis Research. 136 (1), 107-124 (2018).</li><li>Suorsa, M., Paakkarinen, V., Aro, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+native+gel+systems+for+separation+of+thylakoid+protein+complexes:+novel+super-+and+mega-complexes.">Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes.</a> Biochemical Journal. 439 (2), 207-214 (2011).</li><li>Rantala, M., Tikkanen, M., Aro, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+characterization+of+hierarchical+megacomplex+formation+in+Arabidopsis+thylakoid+membrane.">Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane.</a> Plant Journal. 92 (5), 951-962 (2017).</li><li>Porra, R. J., Thompson, W. E., Kriedemann, P. 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A successful thylakoid solubilization and native gel run will result in the resolution of multiple distinct visible green bands on the gel without significant distortion or smearing of the bands. Overloading the gel, a high detergent concentration, an incorrect sample pH, undissolved material, running the gel too rapidly or at too high a temperature, and an improperly poured gel are all factors that may contribute to poorly resolved thylakoid complexes. While optimizing the conditions of the gel itself (e.g., ac...

Photosynthetic organisms must constantly adjust their physiology to changing environmental conditions to maximize their productivity and successfully compete with neighbors1. This is especially true of the machinery responsible for the light reactions of photosynthesis, as ambient light conditions can fluctuate by three orders of magnitude between shadows and full sunlight. Additionally, environmental factors such as drought, cold, or heat stress can reduce the availability of carbon dioxide for carbon fixation, which is the natural electron sink for the products of the light reactions. Plants must, therefore, harvest and utilize solar radiation as efficiently as possible while retaining the ability to dissipate excess light energy as necessary. While photooxidative damage still occurs routinely under all light conditions2,3, failure to manage absorbed excitation energy successfully can lead to the catastrophic cell damage and death. Several adaptive mechanisms exist that allow the photosynthetic apparatus to be tuned both to changes in prevailing environmental conditions and to transient fluctuations (i.e., over both long and short timescales)4. These include the long-term response (LTR) and non-photochemical quenching (NPQ). NPQ is itself considered to encompass at least three other component phenomena, including state transitions (qT), rapidly inducible energy quenching (qE), and photoinhibition (qI)5.
These processes were originally observed and defined largely in terms of spectroscopic phenomena [e.g., NPQ refers to a drop in observed chlorophyll fluorescence (quenching of chlorophyll fluorescence) that is not due to an increase in the rate of photochemistry]6. The term &#34;state transitions&#34; similarly refers to the observed change in the relative amount of fluorescence from PSI and PSII7. While the spectroscopic techniques that have made enumeration of these phenomena possible [in particular, pulse amplitude modulated (PAM) fluorescence spectroscopy] and continue to be a vital means for observing and dissecting photosynthetic processes in vivo, a great deal of biochemistry is required to elucidate the mechanisms underlying these spectroscopic observations. State transitions, for instance, involves a phosphorylation/dephosphorylation cycle of the LHCII proteins by the STN7 kinase and TAP38/PPH1 phosphatase, respectively8,9,10. This cycle adjusts the physical distribution of the LHCII antenna between the two photosystems by moving a portion of LHCII trimers from PSII to PSI, thereby changing the absorption cross section of the photosystems11,12. The qE component of NPQ rapidly converts excess excitation energy into heat through the actions of the violaxanthin/zeaxanthin epoxidation/de-epoxidation cycle and the PsbS protein. The exact role of PsbS in this process is still not fully understood13. The qI component of NPQ, photoinhibition, is generally ascribed to damage to the D1 protein of PSII. Restoration of full photosynthetic competence requires an elaborate repair process to fix damaged PSII photocenters. The PSII repair cycle involves the migration of PSII complexes out of the granal stacks, dismantling of the complexes, replacement of damaged D1 proteins, reassembly of the PSII complexes, and movement of PSII complexes back into the granal stacks14. The exact nature of photoinhibition and PSII photodamage remains a subject of intense scrutiny15.
The difficulty in studying phenomena like state transitions or PSII repair arises in part from the fact that there is not one simple way to visualize the mechanics of complex biochemical systems. The classic biochemical approach to understanding a process is to first separate its components so that they can be characterized in isolation. Native gel electrophoresis arose from successful efforts in the 1980s to separate and characterize the photosystem complexes from the thylakoid membranes with more preparative methods (namely sucrose gradient centrifugation and chromatography)16. The detergent systems developed to gently solubilize the native complexes from the thylakoid membranes were soon adapted to electrophoretic separation methods, most notably by Allen and Staehelin17 and and Peter and Thornber18, giving rise to native green gel electrophoresis. While representing only one out of a variety of techniques in the experimental arsenal, native PAGE has a number of attractive characteristics that have made it a widely employed method in photosynthesis research. Native PAGE is relatively fast and simple, requiring little specialized equipment, while providing high resolution separation of a large number of thylakoid complexes simultaneously. This makes native PAGE a convenient tool for studying thylakoid dynamics and, when combined with standard PAGE in the second dimension as well as a variety of detergent and buffer systems, a versatile system for finding and characterizing new thylakoid complexes.
That being said, native green gels have had a reputation for being an unreliable technique, especially in inexperienced hands, as it is easy to produce poor results consisting of fuzzy, smeary gels with few bands. This problem was solved, in part, with the introduction of blue-native PAGE19. The use of coommassie dye in the BN buffer system makes protein separation more robust. Therefore, BN-PAGE is often an easier and more reliable technique for a relative novice to set up and can provide high resolution separations of thylakoid complexes. For these reasons, BN-PAGE has become the method of choice for most work of this field. While BN-PAGE is generally slower to run than green gel electrophoresis, its main drawback is that the coommassie dye staining interferes with the identification of faint chlorophyll-containing bands, while also making downstream spectroscopic characterization problematic.
The biochemical information provided by native gels and 2D SDS-PAGE can be greatly strengthened when combined with data from spectroscopic techniques. Regardless of the system employed, a central problem with using native gels to identify complexes is that the identification can always be challenged (i.e., the proteins found in a band could always represent comigrating complexes or components, rather than a single physiologically authentic complex). Spectroscopic characterization provides biophysical information about the pigments in green gel bands and can be used to determine what types of complexes they are likely to contain. Chlorophyll fluorescence is especially useful in this regard due to the often dramatically different spectra and fluorescence lifetimes that are characteristic of different photosynthetic pigment-protein complexes. While simple steady-state 77K fluorescence spectra have historically been useful in confirming the identities of native gel complexes, modern time-correlated single photon counting (TCSPC) can provide much more information. TCSPC allows not only the characterization of complexes based on fluorescence lifetimes, but also makes possible the detailed description of energy transfer between spectral components within a complex. This kind of characterization is becoming increasingly necessary as the use of various native gel systems spreads and new putative complexes are discovered, allowing the identification of protein complexes to be better authenticated and providing new biophysical information about how these complexes work.
In this paper we provide a method that allows those having little or no experience with native gel electrophoresis to achieve high quality resolution of native thylakoid complexes for the purpose of investigating the mechanics of the light reactions of photosynthesis. This basic technique can then be augmented at the experimenter&#39;s discretion to improve results or extend applicability to other species. We then describe the process for subjecting native green gel bands to TCSPC, as well as some steps for basic analysis and presentation of the data provided by the technique. The coupling of native gel electrophoresis with TCSPC analysis extends the utility of these gel systems by providing authentication and biophysical characterization of protein complexes within the bands. The green gel system described here is based on that developed by Allen and Staehelin17 with some modifications and is the same as that used in Schwarz et al.20. This system is one of many but has specific features that are useful for this methodology. It is rapid enough so that thylakoid isolation, gel electrophoresis, and TCSPC analysis convenient can be performed in one day, obviating potential problems of sample storage and degradation. We also find that this method is robust in the hands of inexperienced users, while still providing results that range from good to superior, depending on the degree of optimization.
It is important to bear in mind that the complexes visualized on a native gel depend on both the detergent and buffer systems used, as well as on the biology of the organism under investigation. Different detergent and buffer systems preferentially separate different kinds of complexes, and a given photosynthetic organism will have different complexes from other organisms, not all of which will be present under any given circumstance. The system described here is particularly suited to the study of PSI megacomplexes, as described in Schwarz et al.20, but it falls on the more destabilizing end of the spectrum for those studying PSII megacomplexes. For a comprehensive study of the various detergent and buffer systems used in native gel electrophoresis of thylakoid proteins, it is recommended to review J&#228;rvi et al.21 and Rantala et al.22.

<table>
	<tbody>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>#648310</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Decyl Maltoside</td>
			<td>Sigma</td>
			<td>D7658</td>
			<td>n-decyl beta d maltopyranoside, not dodecyl maltoside or alpha decyl maltoside</td>
		</tr>
		<tr>
			<td>Octyl Glucoside</td>
			<td>Sigma</td>
			<td>O8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>BioRad</td>
			<td>161-0148</td>
			<td>37.5/1 C 40% solution</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>BioRad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>BioRad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of spinach thylakoid protein complexes by native green gel electrophoresis and band characterization using time-correlated single photon counting

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and organization of these complexes is highly dynamic on both long and short time scales due to processes that adapt photosynthesis to changing environmental conditions (i.e., non-photochemical quenching, state transitions, and the long-term response). Historically, these processes have been described spectroscopically in terms of changes in chlorophyll fluorescence, and spectroscopy remains a vital method for monitoring photosynthetic parameters. There are a limited number of ways in which the underlying protein complex dynamics can be visualized. Here we describe a fast and simple method for the high-resolution separation and visualization of thylakoid complexes, native green gel electrophoresis. This method is coupled with time-correlated single photon counting for detailed characterization of the chlorophyll fluorescence properties of bands separated on the green gel.

Representative results for green gel electrophoresis are presented in Figure 1. Lane 1 provides an example of ideal results for green gel electrophoresis of spinach thylakoids, in which a maximum number of clear, sharp green bands are visible. These results are somewhat atypical, in part because not all of the bands seen in lane 1 are normally present in a given sample. Additional sample cleanup, in the form of chloroplast isolation before thylakoid solubiliz...

Watch this Scientific Journal Video about Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting a

Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting

Here we present a protocol to separate solubilized thylakoid complexes by Native Green Gel electrophoresis. Green gel bands are subsequently characterized by Time Correlated Single Photon Counting (TCSPC) and basic steps for data analysis are provided.

The light reactions of photosynthesis are carried out by a series of pigmented protein complexes in the thylakoid membranes. The stoichiometry and ...

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