2. Materials and Methods
2.3 Standard techniques
2.3.1 Absorbance spectroscopy
Absorbance spectra were taken from the samples specified in the protocol, at room temperature using a Cary 50 spectrophotometer between the wavelengths specified in the graphs.
2.3.2 Fluorescence emission spectroscopy
77K fluorescence was performed in an Oxford Scientific OptistatDN liquid nitrogen-cooled cryostat (Oxford instruments). Minor adjustments to the sample location in the holder etc were performed prior to attaining the 77k fluorescence spectra shown. Fluorescence emission spectra were obtained using a Fluorolog®-3 spectrometer using a Xenon light source.
2.3.3 Chlorophyll quantification assay
Chlorophyll concentration of samples was measured by adding 4ul sample to 2ml of 80% acetone to liberate chlorophylls, followed by vortexing and spinning in a benchtop centrifuge at 13k for 2 minutes to pellet the apoprotein. Following this an absorbance spectrum between 800 and 500nm was taken, and the chlorophyll concentration and a/b ratio was obtained using a custom-made python script, utilising the following equation:
A646corrected = A646 – A750
A663corrected = A663 – A750
[Chl a] = (12.25 x A646 corrected)– (2.55 x A646corrected)
[Chl b] = (20.31 x A646corrected) – (4.91 x A663corrected)
Total chlorophyll concentration = [Chl a] + [Chl b] Chlorophyll ratio = [Chl a] / [Chl b]
2.3.4 Sucrose gradients
Continuous sucrose gradients were generated by using two sucrose solutions in a Hoefer™ SG Series Gradient Maker, using a peristatic pump to pour the gradient, and load the sample. Stepwise sucrose gradients were formed by layering each decreasing sucrose concentration from bottom to top. Centrifugation was performed at 4°C at the relative centrifugal force given in the protocol. Fractions
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were removed from a sucrose gradient using a needle connected to a peristatic pump. The sucrose concentration of these fractions was measured using a refractometer. The buffer used for the sucrose gradient is given in the protocols.
2.3.5 Columns
Unless stated otherwise: Columns requiring a continuous gradient for elution (eg. anion columns) were run in GE healthcare AKTAprime plus pump system, recording the elution profile for the absorbance at 280nm (aromatic residues) and collecting 1ml samples automatically. Columns utilising steps for elution (eg. Ni2+ charged IMAC columns) were simply connected to a peristatic pump and
pumped directly onto the column, with flow through from the column being collected in 1ml fractions.
2.3.5.1 Anion exchange
Columns were either bought pre-packed 5ml columns, or self-packed columns of resin for either DEAE anion resin (HiTrap™ DEAE FF, DEAE Sepharose Fast Flow anion exchange chromatography resin) for weak binding, or Q anion resins (HiTrap™ Q FF, Q Sepharose Fast Flow) for stronger binding, from GE Lifesciences. Columns were always equilibrated in 5mM NaCl, and the gradient ran according to the protocol.
2.3.5.2 Ni
2+charged IMAC columns
Columns were either prepacked 5ml columns (GE Lifesciences HisTrap FF) or self-packed chelating resin (GE Lifesciences Chelating Sepharose™ Fast Flow) which was then charged using 100mM NiSO4.
Following this it was run with the buffers given in the protocol as described by 2.3.5.
2.3.5.3 Hydroxyapatite column
The Bio-rad Bio-Scale™ Mini CHT™ Ceramic Hydroxyapatite Cartridges type I 5ml prepacked columns were initially run in 400mM sodium phosphate pH 7.5 for 10 column volumes to equilibrate the column for the first use. Following this they were equilibrated and run in the buffer given in the protocol.
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2.3.5.4 Desalting column
GE Lifesciences HiTrap™ Desalting columns were run on the AKTAprime plus pump system and equilibrated with 20 column volumes of the buffer given in the protocol. Following this the sample was loaded and the columns were run at 3ml/min, collecting 1ml fractions.
2.3.5.5 Gel filtration
FPLC gel filtration column (HiLoad 16/600 Superdex™ 200 prep grade) was equilibrated in the buffer given in the protocol overnight at 0.1ml/min utilising the AKTAprime plus pump system. Following loading of the sample onto the column the column was run at 0.2ml/min overnight, taking 2 ml fractions.
2.3.6 Protein analysis methods
2.3.6.1 SDS PAGE
Protein samples were diluted appropriately to a final volume of 15µl, and then incubated with 5µl of NuPAGE® LDS sample buffer 4X loading dye for 20 minutes. The entire 20µl sample was then loaded into pre-cast NuPAGE® 12% Bis-Tris polyacrylamide gels (NuPAGE® system). 10µl of precision plus protein™ unstained standards (Bio-Rad) were loaded into each end of the gel if enough lanes remained. Gels were run at 180V for 50 minutes in NuPAGE® MES SDS Running buffer. Following this, gels were stained in Coomassie Brilliant Blue R250 (Or Bio-Rad™ SYPRO Ruby Protein Gel Stain for figure 5.2) for 20 minutes, followed by de-staining in MQ water overnight to attain a good contrast. Gels were then imaged in an Amersham Imager 600.
2.3.6.2 Immunoblotting (Western blot)
SDS-PAGE gels were run as stated in section 2.3.6.1, however Precision Plus Protein™ Prestained Standards Dual colour (Bio-Rad) was loaded in place of the unstained standard, and gels were not stained following being run. Instead, gels were transferred at 300mA for 1 hour onto an Invitrolon™ PVDF membrane, in 20% methanol, 200mM glycine, 25mM Tris 0.3% SDS. Following this these were blocked with 5% dried milk powder in washing solution (10mM Tris pH 7.5, 150mM NaCl, 0.05% Tween 20) for 30 minutes. The membranes were washed 3 x for 15 minutes in washing solution, then incubated overnight in an appropriate concentration (usually 2000-5000x dilution) of the primary
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antibody at 4°C. After this, membranes were washed 3x in washing buffer for 15 minutes, then incubated in a 1000x dilution of anti-rabbit secondary antibody for 40 minutes. This was followed again by 3x15 minutes in washing buffer, followed by development of the membrane using the CYANAGEN WESTSTAR SUN development kit for western blot. This was then imaged using the chemiluminescence mode on the Amersham Imager 600. All antibodies were purchased from Agrisera™.
2.3.6.3 Blue Native PAGE
Protein samples were diluted appropriately to a final volume of 15µl, and then incubated with 5µl of blue native loading mix (125mM Tris pH 6.8, 30%(v/v) glycerol, 0.01% (w/v) bromophenol blue). The entire 20µl sample was then loaded into pre-cast NaivePAGE™ 4-16% Bis-Tris Gels (NuPAGE® system). Gels were run at 180V for 50 minutes in NuPAGE® NativePAGE running buffer with a 20x dilution of NativePAGE™ cathode buffer additive. Following this, gels were de-stained in MQ water overnight to attain a good contrast. Gels were then imaged in an Amersham Imager 600.
2.3.7 SATP treatment of Plastocyanin and confirmation
Treatment of plastocyanin with Pierce™ SATP (N-succinimidyl-S-acetylthiopropionate) and the subsequent deacetylation to expose the thiol group was performed as stated in the user guide on the ThermoFisher Scientific website. To confirm this SATP had attached correctly, following deacetylation the SATP treated plastocyanin was incubated with ca.30µM of Alexa Fluor™ 750 C5 Maleimide (ThermoFisher Scientific) in 20mM HEPES pH 7.4, 20mM NaCl for 1 hour. Following this it was run down a desalting column to observe in the fractions whether the dye would elute with the protein or separately.
2.3.8 Lowry assay
A detergent compatible kit was used for determination of protein concentration. Details of the protocol can be found on the Bio-Rad® website under DC™ Protein assay. No deviations were made from the protocol.
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2.3.9 Kinetic absorbance measurements
Kinetic traces were recorded on an Olis RSM 1000 UV/Vis rapid scanning spectrophotometer, which facilitated a scan rate of 64 spectra per second, for a spectral width of 300nm centred on the wavelength given in the protocol.
2.3.9.1 Cytochrome b
6f stopped-flow
Two mixtures (A and B) were generated for stopped-flow experiments on cytb6f : Pc. Mixture A
comprised of 1.25mM decylplastoquinol, which has been reduced via the method seen in Trumpower and Edwards, 1979. Following this reduction and extraction, the decylplastoquinol powder was dissolved in DMSO to generate a stock, and mixture A was made from this stock. Mixture B comprised of 62.5 µM Pc, and 230nM cytb6f. Both A and B were in a buffer of 20mM HEPES pH 7.4, 50mM NaCl,
0.2mM tPCC-α-M. A and B were mixed in a ratio of 1:4 in a USA stopped-flow accessory for the Olis, giving end concentrations of 185nM cytb6f, 50 µM Pc, 250 µM decylplastoquinol. Control
measurements were performed by omitting either decylplastoquinol (but still pipetting the same amount of DMSO into the sample as would come from the stock) or cytb6f (to measure the background
rate of ET from decylplastoquinol to Pc). The absorbance changes were measured as stated in 2.3.9, with the centre of the recorded spectra at 600nm, and the change in Pc[Ox] was measured using an extinction coefficient of 4.7mM-1 cm-1 at 597nm.
2.3.9.2 PSI flash excitation
The PSI mixture measured comprised of 215µM Pc, 500µM methyl viologen and 60nM PSI (when present), in 50mM HEPES pH 7.4, 50mM NaCl, 5mM MgCl2, 0.03% (w/v) βDDM. Illumination of the PSI
mixture was carried out by a 0.5W 420nm Thor labs LED at 70% power. Filters at 550nm were used to minimise the noise from illumination. The LED power was controlled by a DC2200 - High-Power 1- Channel LED Driver, and the signal for the 10 second illumination was provided by a custom-built Arduino setup. The absorbance changes were measured as stated in 2.3.9, with the centre of the recorded spectra at 600nm, and the change in Pc[Ox] was measured using an extinction coefficient of 4.7mM-1 cm-1 at 597nm.
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2.3.9.3 RC-LH1 flash excitation
The RC-LH1 mixture for both WT and QE(L264) mutants comprised of 20 µM cyt c26His (Pre-reduced via
treatment with ascorbate then desalted into 50mM HEPES, 50mM NaCl, 0.03% (w/v) βDDM (kinetic buffer) to remove ascorbate, 2.3.5.4), 10nM RC12His-LH1 and 50µM coenzyme Q
0 all in kinetic buffer.
Excitation of the mixture was provided by a 0.5W 880nm Thor labs LED at 80% power. Filters of 730nm were used to minimise noise from illumination. The LED power was controlled by DC2200 - High-Power 1-Channel LED Driver, and the signal for the 10 second illumination was provided by a custom-built Arduino setup. The absorbance changes were measured as stated in 2.3.9, with the centre of the recorded spectra at 550nm. The changes in cyt c2[Red] were followed using an extinction coefficient
of 30.8mM-1cm-1 at 550nm.
2.3.10 Electron microscopy
The brief electron microscopy in this thesis was performed by L.Malone, D. Swainsbury and P Qian from the Department of Molecular Biology and Biotechnology at the University of Sheffield.