Purification of endogenous middle module
Δ yeast strain (BY4741; MATa
::clonNAT) was obtained from Stephan Jellbauer (Gene Center Munich) and C-terminal TAP-tags were introduced on Med7, Med15 or Med18, respectively, using a kanMX4-marker by means of vector pYM13 (30
). Yeast cultures were cultivated and the protein complexes purified using tandem affinity purification as described (15
Preparation of recombinant middle module
Monocistronic vectors were cloned by standard procedures. Bi- or tricistronic vectors were generally created by introducing additional ribosomal binding sites by means of PCR into pET- and pCDF-vectors (Novagen) as described (13
) and illustrated in . A detailed listing of all vectors used and created for this study can be found in Supplementary Table S1
. For expression, E. coli
BL21 (DE3) RIL cells (Stratagene) were transformed with one to three plasmids and grown in LB medium at 37°C to an optical density at 600 nm (OD600
) of 0.6. Expression was induced with 0.5 mM IPTG for 16 h at 18°C. Cells were lysed using a high-pressure homogenizer (Avestin) (large-scale protein purification) or by sonication (Branson) (small-scale purification).
Figure 2. Recombinant Mediator middle module. (A) Recombinant coexpression of multisubunit protein complexes in E. coli is possible by using multicistronic expression vectors, several compatible vectors cotransformed into E. coli, vectors with several promoters, (more ...)
The quaternary complex Med7/10/21/31-His6 was expressed from two plasmids encoding Med10, Med7 and Med21 in a tricistronic pET21b vector (pSB104) and Med31-His6 in a pET24d vector (pSB102). Four liters of E. coli culture were harvested and lysed in buffer A (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM β-mercaptoethanol) containing protease inhibitors. After centrifugation, imidazole was added to a final concentration of 20 mM to the supernatant and loaded onto a 3 ml Ni-NTA gravity flow column (Qiagen) equilibrated with buffer A containing 20 mM imidazole. The column was washed with 20 column volumes (CVs) of buffer A containing 20 mM imidazole and eluted with buffer A containing 200 mM imidazole. The 4-subunit middle module was further purified by anion exchange and gel filtration chromatography.
The Med4/9 complex was coexpressed from a bicistronic pET21 vector (pSB118) using 2 l of E. coli culture. Cells were harvested and lysed in buffer B (20 mM Tris pH 8.0, 150 mM NaCl, 10 mM β-mercaptoethanol) containing protease inhibitors. Insoluble cell debris were removed by centrifugation and the proteins purified by ammonium sulfate precipitation and anion exchange chromatography. Ammonium sulfate precipitation was achieved by the gradual addition of saturated (20°C) ammonium sulfate solution up to 30% (v/v). After centrifugation, the pellet was resuspended and purified on a MonoQ 10/100 column (GE Healthcare) in buffer C (50 mM Tris pH 8.0, 100 mM NaCl, 10 mM β-mercaptoethanol) using a linear gradient of 20 column volumes (CVs) from 100 mM to 1 M NaCl. A six-subunit middle module comprising Med4/7/9/10/21/31 was obtained by assembling the two purified subcomplexes. Assembly was performed at 20°C on a rotating wheel with a 1.3 molar excess of Med4/9. Contaminants, excess Med4/9 and unassembled 4-subunit middle module were separated using anion exchange chromatography (buffer C, 20 CV from 100 mM to 1 M NaCl). Following concentration in a 100 kDa MWCO spin concentrator (Amicon Ultra, Millipore), the 6-subunit middle module was purified to homogeneity by gel filtration chromatography using a Superose 6 size exclusion column (GE Healthcare) with buffer A.
The complete middle module comprising Med1/4/7/9/10/21/31 was expressed from the cotransformed vectors pSB104 (encoding Med10, Med7 and Med21), pTK26 (encoding His6-Med31) and pTK114 (encoding StrepII-tagged Med1, Med9 and Med4). After cotransformation of two vectors, cells were made competent again in order to transform the third vector. Six liters of E. coli culture were harvested and lysed in buffer D [50 mM Tris pH 8.0, 150 mM NaCl, 10% (v/v) glycerol, 10 mM β-mercaptoethanol] containing protease inhibitors. After centrifugation, the complex was purified on 3 × 1 ml Strep‐Tactin MacroPrep (IBA) gravity flow columns according to the manufacturer’s instructions. Following elution by d‐Desthiobiotin (IBA) addition, the sample was concentrated in a 100-kDa MWCO spin concentrator and subjected to gel filtration using a Superose 6 size-exclusion column (GE Healthcare) with buffer A.
Native mass spectrometry
The buffer exchange for the native MS analysis was performed using 10 kDa cut-off membrane spin columns (Millipore, England) by six sequential concentration/dilution cycles against 200 mM ammonium acetate pH 6.8. The concentration for the analysis was 5 µM (assuming intact protein complexes). Analysis of the intact protein complex, as well as the n-propanol and DMSO measurements were carried out on a LCT mass spectrometer (Waters, UK). Needle voltage was set to 1250 V, cone voltage varied between 100 and 150 V. Tandem mass spectrometry was performed on a modified Q-ToF; here, needle voltage was set to 1300 V and cone to 150 V. For further details see ref. 31 and references within. Ion mobility mass spectrometry (IM-MS) was carried out on a Synapt HDMS (Waters, UK) (32
). The source pressure was set to 6.9 mbar, the pressure in the trap was 3.5 × 10−2
mbar, 0.7 mbar in the ion mobility separation (IMS) cell and 2 × 10−6
mbar in the ToF. The wave height in the IMS cell was fixed on 11.3 V and the wave velocity set to 250 m/s. The gas used in the trap was xenon and nitrogen in the IMS cell. Needle voltage was 1200 V and cone voltage 150 V. The bias value was set to 20 V, trap and transfer collision energy to 12 V. Cross-section calculations were done as described by ref. 33. The average volume of global proteins and protein complexes was calculated according to ref. 34.
Limited proteolysis analyses
Limited proteolysis time courses were performed to identify durations suitable for obtaining medium-size fragments. Digests were performed using 20–50 µg protein complex with 0.2 µg of chymotrypsin (Sigma C3142) in buffer A supplemented with 4 µM CaCl2 by incubation at 37°C for 1–60 min. The reactions were stopped by the addition of SDS sample buffer and were heated immediately to 95°C for 10 min. For experiments depicted in , purified 3-, 4- and 6-subunit middle module complexes were subjected to limited proteolysis for 10 min using chymotrypsin and after stopping the reaction by addition of a protease inhibitor mixture, loaded onto a Superose 6 gel filtration column (GE Healthcare). For proteolysis, 1 µg of sample was used for 3- and 4-subunit, and 2 µg for the 6-subunit middle module. Bands of interest in individual peaks were analyzed on SDS-PAGE after TCA precipitation by MS and Edman-sequencing with a Procise 491 sequencer (Applied Biosystems) following transfer to PVDF membranes.
Figure 4. Limited proteolysis analysis of Mediator middle module. (A) Recombinant complexes were subjected to limited proteolysis by chymotrypsin after identifying suitable digestion durations using time courses. The samples were subjected to gel filtration, the (more ...)
Coexpression and copurification pull-down assays
Coexpression was performed as described above using 50 ml cultures for StrepII-affinity purifications or 7 ml cultures for Ni-NTA purifications. Cell lysates were clarified by centrifugation and copurification pull-down assays performed in batch. StrepII-affinity purifications were performed using 25 µl of Strep‐Tactin MacroPrep (IBA). The clarified lysates were incubated with the beads at 4°C on a rotating wheel for 1 h. Beads were washed with 3 × 1 ml of buffer A and the samples eluted by d-Desthiobiotin. Ni-NTA purifications were performed using 40 µl of MagneHis beads (Promega). The clarified lysates were incubated with the beads at 4°C on a rotating wheel for 15 min. Beads were washed with 2 × 1 ml of buffer A and the samples eluted by 400 mM Imidazole in buffer A. Samples were analyzed on SDS-PAGE.
Small-angle X-ray scattering
Small-angle X-ray scattering (SAXS) data were collected at the X33-Beamline (EMBL/DESY, Hamburg, Germany). Scattering patterns from 6-subunit Mediator middle module solutions comprising Med4/7/9/10/21/31 were measured in buffer A at 5 mg/ml two times in order to exclude potential radiation damage errors. The ATSAS software package (35
) was used for data processing.