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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 conditions^2,3, 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 "state transitions" 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, respectively^8,9,10. 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 photosystems^11,12. 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'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 Järvi et al.²¹ and Rantala et al.²².

Protokół

1. Stock Solutions Preparation for Pouring Native Green Gels

1. 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.
2. 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.
3. Store these buffers at 4 °C to prevent the growth of mold.
   Note: The buffers are stable for months at 4 °C, so preparation of 100-200 mL of each buffer for continued use is recommended.
4. Prepare 1 L of 10x running buffer containing 250 mM Tris HCl pH 8.3, 1.92 M glycine, and 1% SDS. Store the 10x running buffer on the benchtop.

2. Stock Solution Preparation for Isolation and Solubilization of Thylakoids

1. Prepare 100 mL of TMK homogenization buffer containing 50 mM Tris buffer (pH 7), 10 mM MgCl₂, and 10 mM KCl, and store at 4 °C.
   Note: This is the main buffer for homogenizing and manipulating thylakoid samples. Alternatively, concentrated stock solutions can be prepared and diluted as necessary (Tris buffer, MgCl₂, and KCl can all be easily stored on the benchtop as 1 M concentrates).
2. Prepare stock solutions of the thylakoid solubilization detergents, B-decyl maltoside (DM) and n-octyl B-d-glucoside (OG), by dissolving each detergent in TMK buffer at 20% w/v. Freeze at -20 °C in 1 mL aliquots.
3. Prepare thylakoid solubilization buffer (SB), which is also the sample loading buffer.
   1. First, make TMK-glycerol buffer by combining 7 mLs of TMK buffer and 3 mLs of glycerol.
   2. To 800 μL of TMK-glycerol buffer, add 100 μL of DM stock solution and 100 μL of OG stock solution. Store this SB working solution, containing 2% DM and 2% OG, frozen at -20° C in 1 mL aliquots.
      Note: Each aliquot can be thawed and refrozen as needed.

3. Pouring Green Mini Gels for Later Use

1. Prepare separate stacking and resolving gel solutions in 15 mL disposable test tubes.
   Note: The volumes provided are sufficient for a single mini gel using 1.5 mm plate spacers.
   1. To make the stacking gel solution, combine 1.25 mL of 4x stacking gel buffer, 0.5 mL of 40% acrylamide stock solution (39:1 C), and 3.25 mL of water to give 5 mL of 4% acrylamide in 1x stacking buffer. To make the resolving gel solution combine 1.875 mL of 4x resolving buffer, 0.94 mL of 40% acrylamide stock solution (39:1 C), and 4.7 mL of water to give 7.5 mL of 5% acrylamide in 1x resolving buffer.
2. Pour the resolving gel.
   1. Add 50 μL of 10% ammonium persulfate (APS) to the resolving gel solution, then add 10 μL of TEMED, cap the tube, and gently invert several times to mix. Immediately pour the gel solution between the gel plates, leaving approximately 1 cm between the top of the resolving gel and bottom of the comb teeth for the stacking gel.
   2. Gently pipette 100% ethanol onto the top of the resolving gel to level the gel.
      Note: If the gel does not set within 15 min, fresh APS solution should be made and/or new TEMED should be used.
   3. After the gel has polymerized (the interface between the gel and the ethanol will be readily visible and will not move when the gel is tipped), pour off the ethanol and blot with an absorbent paper.
3. Pour the stacking gel.
   1. Add 25 μL of 10% APS to the stacking gel solution, add 5 μL of TEMED, then cap and invert to mix in the same manner as the resolving gel. Pour the gel solution on top of the resolving gel until the space between the plates is completely filled and insert a 10-well comb.
      Note: When ready to be used, remove the comb from the gel and rinse the wells with water, making sure that the wells are straight and unobstructed by gel. The gel can be stored with the comb in place at 4 °C for at least several days.

4. Isolation of Crude Thylakoid Membranes from Spinach Leaves

Note: All steps should be carried out on ice using pre-chilled equipment and buffers. Dim lighting is also recommended. Depending on the experimenter's discretion and the biological processes under study, protease and/or phosphatase inhibitors should be added fresh to TMK buffers before homogenization.

1. Completely homogenize spinach leaves in TMK buffer with a glass Dounce homogenizer.
   Note: Approximately 1 to 2 mL of buffer is normally sufficient for a small baby spinach leaf. A single baby spinach leaf can generally provide enough material to load several wells on a 1.5 mm mini gel.
2. Filter the crude leaf homogenate to remove insoluble debris.
   1. To make a simple filtering device, cut a delicate task wipe in half and fold it into quarters. Pack the delicate task wipe into the bottom of a 5 mL disposable syringe and pre-wet the wipe with TMK buffer.
   2. Use the syringe plunger to press excess buffer out of the delicate task wipe and be sure that the wipe filter is pressed firmly to the bottom of the syringe after the plunger is removed.
   3. Pipette the leaf homogenate onto the center of the wipe filter and use the plunger to pass the homogenate through the filter. Collect the filtered homogenate in a 1.5 mL centrifuge tube.
3. Centrifuge the homogenate at 5,000 x g for 10 min at 4° C to pellet insoluble material, including thylakoid membranes. Discard the supernatant and resuspend the pellet in 1 mL of TMK buffer.
4. Normalize the amount of chlorophyll in each sample by adjusting the volume of each resuspended thylakoid sample so that each sample contains the same total amount of chlorophyll, as described below.
   Note: This will allow each sample to be solubilized in the same volume of detergent and minimizes variability in solubilization due to differences in pellet volume.
   1. To extract chlorophyll from each sample of resuspended thylakoid membranes, take a 50 μL aliquot in a 1.5 mL microcentrifuge tube and add 950 μL of methanol to it. Cap the tube and mix by inverting several times.
   2. Centrifuge the methanol/chlorophyll extract at 10,000 x g for 10 min to pellet precipitated proteins.
   3. Determine the chlorophyll concentration of the pigment containing supernatant according to Porra et al.²³. Take absorbance readings at 652 and 665 nm using a spectrophotometer and a 1 cm cuvette. Determine total chlorophyll concentration using the equation below:
      Chls a + b (μg/mL) = 22.12 (Abs 652 nm) + 2.71 (Abs 665 nm)
   4. Using chlorophyll concentration measurements as a guide, remove and discard some volume from each sample, as necessary, so that each tube contains the same total amount of chlorophyll.
5. Re-pellet thylakoid membranes by centrifugation at 5,000 x g for 10 min. Remove and discard the supernatant. Be careful to remove all supernatant without aspirating any of the pellet.

5. Solubilization of Thylakoid Membranes for Loading onto Native Gels

1. Thaw an aliquot of TMK 30% glycerol detergent solution (SB) and invert several times to mix. Keep it on ice.
2. Dissolve the thylakoid pellet by adding the appropriate volume of SB to give a chlorophyll concentration of 1 mg/mL.
   Note: This concentration is a starting point for finding the optimal solubilization conditions, which must be determined empirically. The chlorophyll concentration must be kept the same between samples to allow valid comparisons to be made.
3. Pipette up and down repeatedly while being careful to avoid frothing of the sample. Keep on ice to allow thylakoid samples to solubilize.
   Note: Solubilize for at least 10 minutes. Solubilization time should be long enough that the difference in solubilization time between samples is minimized.
   Example: If 3 minutes are required to solubilize all samples, then approximately 30 minutes should be allowed for solubilization.
4. Centrifuge solubilized thylakoids at 10,000 x g at 4 °C to pellet insoluble material.
   Note: Solubilized thylakoid samples are stable on ice for hours, but storage at -70 °C should be avoided, as freeze-thaw cycles can result in a loss of megacomplex bands.

6. Separation of Solubilized Thylakoid Proteins by Native Gel Electrophoresis

1. Load the solubilized thylakoid supernatant prepared in step 5.4 directly onto the native gel prepared earlier. For a 1.5 mm gel, load 15 μL of solubilized thylakoid per well.
2. Run the native green gel in essentially the same manner as SDS-PAGE gels using 1x running buffer. Run the gel at 100 V and place the entire gel tank on wet ice for the duration of the run to mitigate resistive heating of the gel.
   Note: The gel should require approximately 2 hours for the free pigment at the migration front to reach the bottom of the gel (Figure 1).

7. Excision of Thylakoid Complex Bands from Native Green Gels

Note: Excising the specific band of interest from the gel is necessary to allow the band to be placed in the beam path and to prevent stray fluorescence from nearby complexes from being collected.

1. Remove the gel from the electrophoresis cell and rinse running buffer off of the gel plates with distilled water. Remove the top plate from the gel and rinse the gel with distilled water.
2. Keep the gel on the bottom glass plate and place the gel and plate on ice. When not in use, keep the gel in the dark and cover with a plastic wrap to prevent it from drying out.
3. With the gel remaining on the glass plate, excise each band of interest when ready for TCSPC analysis. Excise bands cleanly with a sharp scalpel or razor blade and take care that the excised band contains no contaminating band material.

8. Collection of Room Temperature Steady-State Fluorescence Spectra

1. For each complex that will be analyzed by TCSPC, a room temperature fluorescence spectrum is taken between 600 and 800 nm using a fluorescence spectrometer.
   Note: The excitation wavelength used to collect this spectrum must match the wavelength used for TCSPC.

9. TCSPC of Green Gel Bands

Note: Refer to Figure 2 for a depiction of the TCSPC setup.

1. Sandwich the gel slice between two glass microscopy slides. Use masking tape, placed on each end of one of the microscopy slides and folded over several times, to create spacers so that the slides can be held together firmly without compressing the gel slice.
   Note: This will create a path for the laser beam to pass between the microscope slides and through the gel slice.
2. Add a small amount of water to the gel slice at the edge of the glass slides to create a smooth interface that will reduce signal scattering.
3. Clamp the gel/slide sandwich in the beam path so that the beam strikes the gel slice through the open edge of the plates, allowing fluorescence emission to be through the side of the glass slides in which the gel is sandwiched, perpendicular to the beam path.
   Note: The excitation wavelength used will depend on the experiment. A wavelength of 435 nm will excite both chlorophyll a and chlorophyll b, while 465 nm will preferentially excite chlorophyll b. In this case, 435 nm was used as the excitation wavelength.
4. Collect 10,000 total data points at regular intervals across the fluorescence emission spectrum for each complex. For example, collect data every 10 nm, starting at 680 nm and ending at 750 nm.
   Note: Prepare multiple gel bands for each complex to be studied so that fresh sample is available in the event that photobleaching prevents adequate signal collection.

10. TCSPC (Data Analysis)

1. For a given complex, first normalize the peak height of each decay curve for all wavelengths collected.
   Note: This step is not necessary for the construction of DAS but allows for decay curves to be overlaid and compared visually with one another as a first inspection of the data.
2. Tail-match each decay curve to the steady state fluorescence spectrum of the complex as described below.
   1. Choose a timepoint after which the decay signal has flattened out, usually after a few nanoseconds.
   2. For each wavelength, normalize the decay curve so that the signal intensity at the selected timepoint is equal to the value of the steady state fluorescence spectrum at that wavelength (i.e., the values of all wavelengths together at the selected timepoint will re-create the steady state fluorescence spectrum).
3. Build decay-associate spectra (DAS) from the tail-matched decay curves, as described below.
   1. Using data points from the tail-matched decay curves construct a series of plots graphing fluorescence intensity vs. wavelength at regular time intervals (e.g., every 10 ps). For example, the DAS at 50 ps is constructed by plotting the value of each decay curve at 50 ps vs. wavelength.
   2. Overlay all of the decay-associated spectra to create a waterfall style plot that shows the decay of the fluorescence spectrum over time.

Wyniki

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...

Dyskusje

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...

Materiały

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