Preparation of specifically labelled purple membranes
1.1 Cultivation of Halobacterium salinarum
Different protocols were followed for the preparation of hydrogenated and deuterated cultures.
1.1.1 Unlabelled cultures of H. salinarum (standard medium)
Cultures of Halobacterium salinarum were described by Oesterhelt & Stoeckenius, 1974 and Oesterhelt & Kripphal, 1983, with some modifications.
Medium for 1 litre of culture:
|
MgSO4 × 7 H2O |
20 g |
|
2 g |
|
|
Na3-citrate × 2 H2O |
3 g |
|
10 g |
|
|
H2O (distilled) |
The pH was adjusted to 7-7.2 and the medium was autoclaved.
Liquid cultures of H. salinarum were cultivated in volumes of 35 ml (100 ml Erlenmeyer flask) and 700 ml (2 l Erlenmeyer), and inoculated with 1 and 35 ml of a liquid culture, respectively. The growth rate of the cells was monitored by light scattering at 600 nm or with a Klett-photometer. The stationary phase was reached after about 7 days in the dark at 37 ºC.
1.1.2 Deuterated cultures of Halobacterium salinarum
Deuterated medium was prepared according to Patzelt et al., 1997. Only fresh D2O was used and the medium was sterilised by filtration through a sterile filter with a pore size of 0.45 mm.
Medium for 1 litre of culture:
|
NaCl (1) |
245 g |
|
MgSO4 (waterfree) |
6.8 g |
|
KCl |
2 g |
|
Tris-D |
1.2 g |
|
deuterated peptone (2) |
5 to 6 g |
|
solution III |
10 ml |
|
solution IV |
100 ml |
|
solution V |
200 ml |
|
D2O |
added to 1 l |
The pD was adjusted to 7.0.
The pD value in D2O is obtained in the following way: pD = pH+0.41 with pH corresponding to the value read with an electrode calibrated in H2O buffers.
The solutions III, IV and V were prepared as followed:
Solution III (additional salts)
|
KH2PO4 |
3 g |
|
K2HPO4.3
H2O |
3 g |
|
KNO3 |
2 g |
|
D2O |
added to 200 ml |
The pD was adjusted to 7.0.
Solution IV (metals)
|
ascorbic acid |
2 g |
|
CuSO4.5 H2O |
5 mg |
|
FeCl2.4 H2O |
230 mg |
|
MnSO4.H2O |
30 mg |
|
ZnSO4.7 H2O |
44 mg |
|
CaCl2.2 H2O |
70 mg |
|
D2O |
added to 10 ml |
For avoiding precipitation it is best to add ascorbic acid at the beginning and CaCl2 as last component.
Solution V (vitamins)
|
thiamine |
|
|
folic acid |
|
|
biotin |
|
|
D2O |
added to 10 ml |
Concentrated NaOD was added until a complete dissolution of all components was reached.
Solutions IV and V were stored at – 20 ºC.
All cultures were grown in Erlenmeyer flasks at 37 °C with shaking at 100 rpm. The bacteria were adapted in several steps to the algae peptone and D2O. First, cultures in D2O and standard medium were inoculated with cells arising from H2O cultures. These cells were then used as inoculum for fully deuterated cultures (medium comprised) in a volume of 35 ml, which served for the inoculation of a 700 ml culture. On average, the stationary phase of the culture growth was reached after 10 to 14 days.
1.1.2.1 Modification for deuterated cultures with H-labelled retinal, methionine and tryptophan
2.5 mM of methionine (Met) and 0.5 mM of tryptophan (Trp) were added to the medium. The retinal negative Jw5-strain was used and hydrogenated retinal, from a stock solution of 10 mM retinal in isopropyl, was added during culture growth. The retinal solution was stored in the dark at –20 ºC.
Special care had to be taken as the hydrogenated retinal had to be added at defined growing steps to ensure the production of Bacteriorhodopsin (BR). 2 µM of retinal was added at six equally spaced steps during the growth process. Once the stationary phase was reached further addition of retinal was avoided.
1.1.3 Preparation of Agar plates
Cell colonies were stored on agar plates. An amount of 15 g of Bacto-Agar was added to one litre of standard medium. About 40 ml of the warm medium was poured into petri dishes. Once, the agar was solidified, 50 ml of a 10-6 diluted liquid culture were spread over a plate and put in a humid environment at 40 ºC. The first purple colonies appeared after several days.
1.2 Isolation of Purple Membranes
Purple Membranes (PM) were prepared with minor modifications as described by Oesterhelt & Stoeckenius, 1974.
The process of PM isolation did not differ for hydrogenated and deuterated cultures. All steps were carried out at room temperature and centrifugation was carried out at 4 ºC.
Cultures were first centrifuged for 1 min at 10,000 g for removal of cell debris, and then cells were harvested by further centrifugation for 15 min at 10,000 g (Sorvall, rotor GS3). The cell pellet was resuspended in 25 ml of basal salt (standard medium without peptone). To avoid excessive viscosity from liberated DNA 1 mg of DNase from bovine pancreas (Fluka) was added. After stirring for several hours at 4 ºC the cells were dialysed (cut-off 12,000 to 14,000 Da) overnight against 10 l of deionised H2O. The purple lysate was centrifuged at 40,000 g (Sorvall, rotor SS34) for 60 min and the red supernatant decanted. The purple sediment was resuspended in H2O, pottered and again centrifuged. These washing steps were repeated until the supernatant became almost colourless. Usually three centrifugation steps were necessary. The final sediment was taken up in a small amount of H2O and layered over a linear 25 to 45% (weight/weight) sucrose density gradient. A 40 to 60% gradient was performed for deuterated PM. Typically, equilibrium was reached after 14 h of centrifugation at 100,000 g and 8 ºC (Beckman, rotor TST28, volume 35 ml). The buoyant density accounted to 1.18 g/cm3 for the unlabelled hydrogenated PM and about 1.26 g/cm3 for the labelled deuterated PM. The purple band was collected and sucrose was removed by dialysis overnight against 10 l H2O. Water was renewed once after a few hours of dialysis.
PM can be considered as pure if the ratio in the UV-Vis absorption spectrum between 280 and 570 nm is smaller than 2.0. The concentration of BR in solution can be calculated as follows:
with: OD570: optical density at 570 nm,
MWBR = 27026 Da, molecular weight of BR,
e57 0= 63000 M-1cm-1, molar extinction coefficient at 570 nm;
L: optical path in cm and
d: dilution factor of the measured sample.
1.3 Reconstitution of PM
Reconstitutions of Purple Membranes were prepared with BR molecules and endogenous lipids. Both components were either hydrogenated or deuterated. A gelfiltration of pre-solubilised PM was performed to obtain delipidated BR. Lipids were extracted from PM, dissolved in n-octyl-b-D glycopyranoside (OG) and mixed in a fixed ratio with delipidated proteins. The reconstitution membranes were finally put on a sucrose density gradient.
1.3.1 Delipidation of PM
The delipidation of PM is based on a protocol from Huang et al., 1980. An aqueous PM suspension was centrifuged for 30 min at 88,000 g and 10 ºC. The pellet was resuspended in 1% (w/v) Triton X-100/10 mM Na-Phosphate buffer, pH=5.6 and stirred for 14-16 h at 34 ºC in the dark. A ratio of 1:4 (weight BR/weight Triton) was necessary for efficient solubilisation. The absorption maximum in the UV-Vis spectrum of solubilised BR shifted to 548 nm. After centrifugation for 1 h at 71,000 g and 10 ºC only the supernatant was kept, its pH adjusted to 8 and sterilised through a Millipore filter (0.45 mm sizepore). The sample was applied to a gelfiltration column with a volume of 177 cm3 (1.5 x 100 cm2). Priorly, the column was packed with Bio-Gel A-0.5m (Biogel) matrix for the separation between delipidated BR, lipid and Triton micelles, and the detergent exchange from Triton X-100 to desoxycholate (DOC) was performed.
The column was eluted in the dark at 4°C with 8 ml/h of 0.01 M Tris-HCl, pH=8/0.15 M NaCl/0.25% DOC. The elution profile of the collected fractions (~1.5 ml) at 280 nm was monitored with an optical detector and a graphical recorder. The absorption maximum of delipidated BR was shifted to a wavelength of 540 nm. A ratio smaller than 1.9 between the absorption at 280 nm and 540 nm holds for Triton free BR (Huang et al., 1980).
Delipidated BR was stored at -20°C to avoid denaturation. The yield of the delipidation process is about 40% of BR from the starting material.
1.3.2 Lipid extraction of endogenous PM lipids
Endogenous lipids were extracted from PM with a CHCl3/MeOH method modified from Bligh & Dyer, 1959. Only clean glassware, rinsed with CHCl3, should be used. The suspension has to be shaken well after any addition of components.
In a separatory funnel salt free PM in 10 mM Citric acid at pH=4.0 was mixed with CHCl3 and MeOH to establish a monophasic mixture of CHCl3/MeOH/H2O in a ratio of 1/2/0.8 (v/v/v)[scm1]. Phase separation was achieved by establishing a final ratio of CHCl3/MeOH/H2O = 2/2/1.8. If no clear phase separation was obtained, the suspension was centrifuged for 5 min at 5,800 g in aluminium or glass bottles. The lower yellow lipid CHCl3 phase was decanted and CHCl3 was added to re-establish the former ratio. This was repeated about three times until the CHCl3 phase was no longer yellow. For removing traces of water Na-sulphate was added to the decanted CHCl3 phase. Finally, the lipid phase was filtered through a folded filter (Faltenfilter) and CHCl3 was evaporated in a rotary evaporator. The yield of the extracted lipids was measured by weighing and found close to 100%.
This protocol can be applied to an extraction on a micro or macro scale. In the case of a micro extraction, glass test tubes were used and the lower CHCl3 phase was withdrawn with a Pasteur pipette after centrifugation in a table centrifuge for 5 min at 3000 rpm. CHCl3 was blown off using N2.
The extracted lipids were stored at –20 °C.
1.3.3 Reconstitution of PM
The procedure for the reconstitution of PM was modified from previously described reconstitutions of halorhodopsin (Bamberg et al., 1993).
Dried lipids were dissolved in 1% (w/v) OG/0.15 M NaCl/0.01 M Tris-HCl, pH=8 to a concentration of 2 mg lipids/ml. A clear translucent yellow solution was obtained by using an ultrasonic bath.
Delipidated BR fractions were concentrated in a stirring cell with a PD10-membrane to a concentration of 1 mgBR/ml. The BR and the lipid suspensions were mixed together in a fixed ratio reproducing the natural density of wild type PM. This ratio was experimentally established and corresponds to 26/2 (mole lipids/mole BR) for the originally most concentrated delipidated BR fractions and 26/2.5 for the less concentrated ones. The ratio in wild type PM is 26/3 indicating that an excess of lipids is required for the reconstitution process. After stirring in the dark for several hours, the mixtures were dialysed (cut-off 10,000 Da, type 20/32) in 60 times their volume for 10 days in the dark at room temperature against 150 mM NaCl/1 mM CaCl2/10 mM Tris, pH=8. The dialysis buffer was renewed daily. The additional 1 mM CaCl2 helped membrane formation.
The purple dialysates were centrifuged for 40 min at 88,000 g at 10 °C (Beckman, rotor 45 TI) and the resuspended sediment is put on a linear density sucrose gradient after homogenisation. A 30 to 50% (w/w) sucrose density-gradient was prepared for the reconstitutions of hydrogenated BR and hydrogenated lipids and for hydrogenated BR and deuterated lipids. Deuterated BR and hydrogenated lipid reconstitutions were packed on a 40% to 60% (w/w) sucrose gradient.