The overall goal of the following experiment is to reconstitute in vitro light harvesting pigment protein complexes of plants or algae. This is achieved by extracting the pigments from spinach leaves or algae cultures, and over expressing the APA prote in e coli. As a second step, the APA prote is mixed with the pigments to obtain a reconstituted complex.
Next, the reconstituted pigment protein complex is purified from the excess pigments and the unfolded protein results are obtained that show similar spectroscopic characteristics of the reconstituted pigment protein complex and the native one based on absorption spectra, fluorescent spectra, and circular deism. The main advantage of this procedure is that it allows us to obtain a relatively high amount of pigment protein complexes, and also to manipulate the complexes using in mutated type protein and pigment mixtures with different compositions demonstrating the procedure will be Alberto na, a grad student in the lab To begin the procedure for extracting total pigment from spinach leaves. Use a blender to homogenize a handful of spinach, leaves approximately 20 grams in 100 milliliters of cold grinding buffer for 20 seconds.
Filter the solution through two layers of nylon cloth with a poor diameter of 20 micrometers and centrifuge the filtrate at 1, 500 Gs for 10 minutes. At four degrees Celsius. Remove the supernatant and add one milliliter of cold wash buffer.
Resuspend the pellet containing the chloroplasts with a soft artist's paintbrush. Once the pellet is resuspended, add 50 milliliters of wash, buffer and centrifuge, a solution at 10, 000 Gs for 10 minutes. At four degrees Celsius, remove the supernatant and gently resuspend the pellet in 50 milliliters of wash buffer centrifuge, a solution at 10, 000 Gs for 10 minutes at four degrees Celsius, remove the supernatant completely from this point onward.
This procedure must be performed in the dark. To avoid pigment oxidation, add approximately 20 milliliters of 80%acetone buffered with sodium carbonate to extract the pigments, leave the solution on ice for 10 minutes. Vortexing occasionally palette the cellular components by centrifugation at 12, 000 Gs for 15 minutes at four degrees Celsius.
Total pigment extraction should result in a white pellet. Collect the snat into a separatory funnel. Add 0.4 volumes of dathyl ether.
Shake vigorously and make sure to open the valves to vent the gas. Add 0.8 volumes of point 33 molar sodium chloride and mix vigorously. Allow approximately 10 minutes for the layers to separate.
The ether phase on top contains the extracted pigments. Remove and discard the clear lower phase. Remove the ether by pouring it from the top of the separatory funnel into a suitable glass container.
Dry the ether by adding a spoonful of granular anhydrous sodium sulfate. Swirl the solution and allow approximately five minutes for the desiccant to absorb water from the ether. When the ether is sufficiently dried.
There should be some free-floating crystals and no clumping of the sodium sulfate. Decant the ether to a new glass container, leaving the sodium sulfate solid behind aliquot the pigments and dry them in a rotary speed vac until the acetone is completely evaporated. Store the dried pigments at negative 80 degrees Celsius.
The extraction of keratinoid from spinach is not shown in this video, but the dried pigments are also stored at negative 80 degrees Celsius. The light harvesting complex or LHC for reconstitution was expressed and purified from e coli in the form of inclusion bodies. For the success of reconstitution, the ratio between pigment proteins and the amount of buffer use is crucial.
This procedure should be performed in dim light. Resuspend 800 micrograms of LHC inclusion bodies in a total of 400 microliters of te. In a two milliliter fuge tube, add 400 microliters of two x reconstitution, buffer and vortex.
Briefly add 0.6 microliters of a 14.8 molar beta mar CAPTA ethanol stock. To obtain a final concentration of 10 millimolar, heat the protein at 98 degrees Celsius for one minute. Vortex briefly and place at room temperature for three minutes.
Resuspend 500 micrograms of total dried chlorophyll pigments, plus 80 micrograms of keratinoid pigments in 30 microliters of 100%ethanol by vigorously vortexing. For one minute. Spin the pigment mix at 15, 800 Gs at four degrees Celsius for about 30 seconds, and confirm that there is no pellet immediately after the spin, add the pigment mix slowly to the cooled protein while vortexing.
Be careful not to vortex too vigorously as the protein can overflow the top of the tube. Continue to vortex for five to 10 seconds and then place the tube on wet ice. Add 94 microliters of 20%octal, beta D glucocide, or og to get a final concentration of 2%Vortex briefly and keep on ice for 10 minutes, add 90 microliters of two molar potassium chloride to get a final concentration of 150 to 200 micro molars.
Vortex briefly and keep on ice for 20 minutes. Spin at 15, 800 GS at four degrees Celsius for 10 minutes. Transfer the SUP natant to a 10 milliliter tube without disturbing the pellet.
Keep the natin cold and protected from light. Begin this procedure by preparing a one milliliter nickel SROs column as described in the protocol text. Add three to four milliliters of OG buffer to the protein sample and load the sample onto the column.
Rinse the column with five milliliters of OG buffer, followed by two milliliters of OG rinse buffer. Elute the bound protein with three milliliters of elu buffer. Collect the green ellu that contains the reconstituted protein.
The sucrose gradient is prepared as described in the protocol text carefully removed from the top of the gradient, the same volume as that of the green fraction alluded from the nickel SROs column. Slowly load the reconstituted sample on top, being careful to avoid disturbing the gradient. Place the tube and a balance into an S SW 41 or SW 60 swinging bucket rotor and spin in an ultracentrifuge at 200, 000 Gs at four degrees Celsius for 18 hours with slow acceleration and stopping without breaks.
When the ultracentrifuge is complete, use forceps to carefully take out the gradient from the tube holder. The green band is a fraction to be collected. The absorption spectrum of CP 24.
A chlorophyll AB binding protein reconstituted in vitro is compared with the spectrum of the native complex. The identical spectra indicate a virtually identical pigment composition and organization. The quality of the reconstituted complex can also be assessed by fluorescence spectroscopy.
Since the pigments emit fluorescence upon excitation at different wavelengths, chlorophyll A at 440 nanometers chlorophyll B at 475 nanometers and zils at 500 nanometers. The fluorescence emissions spectra of a reconstituted CP 24 wildtype complex and normalized to the maximum show efficient energy transfer from chlorophyll B and XANTHE fills to chlorophyll A.The presence of chlorophyll B not coordinated to the protein can be recognized by an additional shoulder around 650 nanometers. The presence of free chlorophyll A instead leads to additional emission around 675 nanometers.
The fluorescent emission spectra at 475 nanometers citation of both the reconstituted and native CP 24 complexes show a single peak at 681 nanometers indicating that the reconstituted complex is correctly folded. Reconstituted CP 24 and the native complex also show very similar circular charism spectra. Single amino acid residues important for the coordination of different pigments can be altered to analyze the properties of individual pigments or assess their contribution to the function and stability of the complex.
Shown here are the absorption spectra of a wild type and mutated CP 29. The green line shows the differences between the two plots When tate, this technique including the SUSE gradient, ultracentrifugation could be done in 24 hour if it is performed properly.
