Protein purification, crosslinking and CD studies
c-DNAs encoding IcmR, IcmQ, Qn(1-66) and Qc(72-191) were cloned into the pQE70 expression vector to create proteins with a C-terminal 6xHis-tag. For co-expression of IcmQ and IcmR, the IcmQ clone was moved to the pET-24a vector, which allowed for selection with kanamycin, and the C-terminal 6xHis-tag was removed in this clone. The expression vectors were transformed separately into XL-1 blue cells or into the BL21 (DE3) cell line for IcmQ (and mutant IcmQs), Qn, Qc(72-191) and IcmR. Cells were grown in 100 ug/ml ampicillin and expression was induced at 37°C with 0.5-1 mM IPTG for ~3 hr. Frozen cell pellets were thawed in 50 mM Tris-HCl or Hepes (pH 8.0), 100 mM NaCl, 10 mM MgCl2 in the presence of a protease cocktail (1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 100 uM chymostatin and 10 uM E64). In addition, 1 uM leupeptin and 125 uM aprotinin were added in some cases. To induce lysis, resuspended cells were treated with lysozyme (0.3 mg/ml of lysate), deoxycholic acid (1.3 mg/ml) and DNase I (30 ug/ml).
To make IcmQ, the high speed lysate (100,000 × g) was bound to Ni-NTA resin and washed extensively with 20 mM Hepes (pH 7.2), 1.0 M NaCl and 20 mM imidazole. The protein was eluted in 20 mM Hepes (pH 7.2), 1.0 M NaCl and 350 mM imidazole and dialyzed extensively to remove the imidazole and excess NaCl against 20 mM Hepes (pH 7.2), 50 mM NaCl (HN buffer) at 4°C overnight. Next, IcmQ was run over a Hi-Trap Q column to remove impurities. IcmQ flows through this column and was then applied to a Hi-Trap S column, where it binds and elutes in two fractions. The first fraction was eluted in 400 mM NaCl and was comprised mostly of IcmQ aggregates, while the second fraction was eluted in 900 mM NaCl and contained IcmQ dimers. The second fraction was dialyzed extensively against HN buffer at 4 °C overnight and stored at -80°C after flash freezing.
Qn57 was purified in a manner similar to IcmQ with the addition of a trypsinization step. After elution from the Hi-Trap S column (900 mM NaCl), 6xHis-Qn66 (which was previously described as Qn (1-72) due to a sequencing error) was treated with trypsin (2000:1 wt/wt) for 2 hr at room temperature. Trypsin was removed with benzamidine beads and the resulting Qn57 dimer was dialyzed overnight against HN buffer at 4 °C. For studies with Qn, gels were fixed with 12% TCA for 30 min before staining with Coomassie blue. The IcmR dimer was purified in the same manner as IcmQ. However, the IcmR dimer did not bind to either the Hi-Trap Q or the Hi-Trap S columns. The flow-through from both columns was collected and dialyzed against HN buffer at 4°C overnight.
The Qcl domain (aa49/50-191) was made by surmounting the Qc folding problem in two steps. First, we engineered two mutations in IcmQ to destabilize the Qn domain (L17D, L39D). This led to selective degradation of Qn by an endogenous bacterial protease. In a second step, two additional point mutations were made in the linker (K57Q, K59Q). These mutations allowed proteolysis to occur at an upstream lysine-arginine pair (aa49/50) and created a larger Qc which retained the linker. This strategy allowed us to make a well-folded, α-helical Qcl domain when over-expressing the IcmQ quadruple mutant in E. coli. The purification of Qcl was similar to IcmQ, with the exception of the final Hi-Trap S column. At this step, Qcl binds the column and elutes in a single fraction in 900 mM NaCl and is not aggregated. The Qcl protein was flash frozen and stored at -80° C.
The co-expressed IcmR-IcmQ complex was made by transforming BL21 (DE3) cells with the pQE-70 vector encoding IcmR with a 6xHis-tag and the pET-24a vector with IcmQ. The cells were grown in the presence of 100 ug/ml ampicillin and 25 ug/ml kanamycin. Protein purification was carried out in a manner similar to IcmQ with the exception of the Hi-Trap S column. In this case, the IcmQ-IcmR complex binds to the column and elutes as a single fraction in 900 mM NaCl. The resulting complex was dialyzed against HN buffer at 4 °C overnight and flash frozen.
For crosslinking studies, proteins were diluted into HN buffer to ~50 uM. DTSSP was used to crosslink IcmQ, IcmR-IcmQ, Qn-(1-66)-6xHis, Rm-Qn and IcmR. Freshly prepared crosslinker was added to the proteins at a final concentration of ~250 uM (1:5 mol/mol ratio). The reaction was allowed to proceed at room temperature for 30 min and was stopped using 1 M Tris HCl, pH 7.5, which was added to a final concentration of 50 mM. The proteins were subjected to SDS-PAGE on 15% gels. For CD studies, AVIV 215 and 62DS spectrometers were used to record far-UV spectra from samples at 0.1 mg/ml in ~5 mM phosphate buffer, pH 7.5 or in phosphate buffer with 100 mM NaCl in a 0.5 mm cell. The CD signal was expressed as molar residue ellipticity.
Membrane association and permeability assays
L-6-phosphatidylcholine (egg PC) was purchased in small ampules (Avanti Polar Lipids, Inc.) and dissolved in chloroform, while 1,2-dimyristoyl–sn-glycero-3-[phospho-rac-(1-glycerol)] (PG) was purchased as a dry powder. The PG was dissolved in chloroform to a concentration of 6 mg/ml. The PC and PG lipids were combined in the ratio of 3:1 (wt/wt), mixed thoroughly, and dried down under nitrogen in 1 mg aliquots. The aliquots were placed under a vacuum overnight to remove trace organics and then stored at −20°C. Vesicles for insertion experiments were made as follows. Lipid aliquots were re-suspended in HN buffer (total volume = 250 ul). Vesicles were made by extrusion through a 0.1 uM porous membrane (Duménil et al., 2004
). Proteins were added to the preformed vesicles (3-4 mg/ml) at protein concentrations of ~1 and 25 uM in HN buffer. For the membrane association assay, a sucrose step gradient was used to separate vesicles from free protein by flotation (Duménil et al., 2004
). In this assay, the protein and vesicle solution was mixed with 60% sucrose (prepared in HN buffer) to a final concentration of 45%. A 30% layer and a 5% layer of sucrose in HN buffer were sequentially layered on top of the 45% layer. Samples were spun for 15 hr at 55000 rpm in an RP-55S swinging bucket rotor at 17°C and equivalent volumes were analyzed by SDS-PAGE with either Coomassie or silver staining, to ascertain the efficiency of protein association with the vesicles.
For the membrane permeation assay, 2 mg of lipids were re-suspended in 20 mM Hepes (pH 7.2), 50 mM NaCl and 10 mM EDTA (HNE buffer, total volume = 500 ul) with the addition of 80 mM calcein. The vesicles were made by extrusion and excess calcein was removed by filtration on a 5 ml G-75 column equilibrated in HNE buffer. To ensure complete removal of free calcein, the vesicles were dialyzed against HNE buffer at 4°C overnight (in the dark). The vesicles were then diluted to a concentration of ~1.2 ug lipid/ml and placed in a cuvette in an ISA SPEX FluoroMax-2 fluorimeter and subjected to constant stirring. Proteins were mixed with vesicles to a concentration of 0.1 uM and the fluorescence emission was monitored at 515 nm as a function of time after excitation at 490 nm.
Crystallographic and mutation studies
Preparation of the Rm-Qn complex, crystallization and structure determination are described in the Supplement
, and includes S_Table 2
, which summarizes the crystallographic data collection and refinements. A description of the methods used to screen for mutants in Qn and in vivo tests for protein stability, DotA secretion and intra-cellular growth in mouse macrophages are also given in the Supplement
. Figures were made with Chimera (Goddard et al., 2005
) and Adobe Photoshop.