SUPPLEMENT: Details of biochemical procedures
Ammonium sulfate treatment changes the monomer to trimer ratio of PS1 which is extracted at
RT after salt-incubation of the thylakoid membranes. The efficiency of this treatment depends
both on the temperature and the salt concentration as shown in Fig. S1.
Fig. S1A
Fig. S1B
Fig. S1C
Fig. S1:
The monomer/trimer ratio can be estimated either from the HIC-HPLC elution profile (A) or
from the PS1 monomer and trimer bands in the sucrose density gradient (B). Both approaches
show an increasing amount of PS1 monomers with increasing ammonium sulfate concentration at
the expense of PS1 trimers, if this treatment is done at 50 °C. Comparison with results obtained at
either 20 or 37 °C (not shown) in a plot (Fig. S1C) indicate that the effect of ammonium sulfate is
dependent on the fluidity status of the membrane, i.e. the liquid-crystalline state (above 37 °C for
Thermosynechococcus elongatus) seems to be the prerequisite for the ability to form extractable
PS1 monomers and reaches saturation at about 0.6 M.
A) HIC-HPLC elution profile of PS1 complexes (POROS 50 OH Applied Biosystems) using
an ammonium sulfate gradient from 1.5 - 0 M according to the recorded conductivity; flow
rate 5 mL min-1 at 10 °C. Incubation of membranes prior to extraction with 0.2 M, 0.4 M and
0.6 M ammonium sulfate at 50 °C, see M&M (elution profiles normalized to PS1 trimer
peak).
B) Separation of thylakoid extract on a linear 0-20% sucrose density gradient, which was
prepared by freezing 20% (w/v) sucrose in buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2,
10 mM CaCl2, 0.03% ß-DM) in 35.8 mL ultracentrifuge tubes at -20 °C, followed by slow
thawing overnight at 4 ˚C. After unfreezing, extracted (acc. to (A)) PS1 (50-100 µL at
0.5 mg/mL Chl in extraction buffer) was added carefully on top of the gradient. Samples were
separated by 18 h ultracentrifugation (82,700 x g, SW 28-rotor, Beckman) at 4 ˚C.
C) PS1 monomer/trimer ratio depending on treatment with various ammonium sulfate
concentrations at 20 °C, 37 °C and 50 °C (followed by extraction at RT and HIC-HPLC
separation; values based on peak areas in the HIC-HPLC elution profile (see A).
(PS1-mono = PS1 monomer, PS1-tri = PS1 trimer)
Monomeric and trimeric PS1 complexes which have been enriched by the hydrophobic
interaction chromatography step (Fig. S1A) can be further purified via ion exchange
chromatography as second step, yielding homogenous PS1 complexes of high purity (see Fig.
S2).
Fig. S2
Fig. S2
Elution profile of PS1 complexes from IEC-HPLC (POROS HQ/M Applied Biosystems) as 2nd
purification step. Peak fractions of monomeric and trimeric PS1 complexes from HIC-HPLC (1st
purification step, see Fig. S1A) have been collected, dialyzed, applied onto the IEC column and
eluted by a 0-200 mM MgSO4-gradient (recorded by conductivity measurement; for details see
M&M). The resulting highly enriched and homogeneous PS1 complexes have then been used for
detailed biochemical and biophysical characterization. (PS1-mono = PS1 monomer; PS1-tri =
PS1 trimer)
In order to find out about possible secondary effects of the pre-treatment of the thylakoid
membrane, PS1 complexes have been extracted under the mildest conditions possible: This
involves omitting the salt treatment and minimizing the amount of detergent used for extraction
(0.1% instead of 0.6% ß-DM) and its exposure time (5 min instead of 15 min at RT). In
summary, this should help to keep the extracted monomer in its native-like structure. Although
under these conditions the absolute amount of extracted PS1 monomer and trimer is considerably
lower (only about 1% of the amount of purified PS1 complexes gained with the standard
procedure including the salt-treatment, see Fig. S1A), the amount of this mildly treated monomer
is sufficient for comparative analysis with salt-treated PS1 monomer (Fig. S1A).
Fig. S3
Fig. S3
HIC-HPLC elution profile of PS1 monomers (POROS 50 OH Applied Biosystems), extracted
without salt treatment by short incubation (5 min RT) at low detergent concentration (0.1 %
ß-DM). (Ammonium sulfate gradient, as determined by conductivity, ranging from 1.5-0 M; flow
rate 5 mL min-1 at 10 °C; for details see M&M). (PS1-mono = PS1 monomers, PS1-tri = PS1
trimers, PS2 = Photosystem 2, Car = Carotenoid, PBS = phycobilisomes)