Purification of Skp, WT-OmpA, TMD-OmpA, FomA and hVDAC1
Skp and wild-type OmpA were purified from E. coli as described [Bulieris et al.
2003]. The construction of the plasmid pET22bB1, expression and isolation of TMD-OmpA were preformed as described previously [Ramakrishnan et al. 2005]. FomA and hVDAC1 were isolated as described [Pocanschi et al. 2006a; Shanmugavadivu et al. 2007].
Purification of OmpG, NalP and YaeT
The ompG gene (signal peptide deleted) was amplified by PCR (60°C annealing temperature) using 50 ng E. coli MG1655 genomic DNA as template and the following primers, TAGGGCCATATGGAGGAAAGGAACGACTGG-3’, and 5’-CCCAAGCTTGCGGCCGCTCAGAACGAGTAATTTACGCCG-3’. The PCR product was cloned into pET28a vector (Novagen) by NdeI/HindIII restriction sites, yielding pET28OmpGm2. Plasmid pET28OmpGm2 was transformed into E. coli BL21 (DE3) (Stratagene) and expressed OmpG protein as inclusion body. 20 ml overnight culture was inoculated into 2 L LB medium. After 3 h, IPTG was added to 0.1 mM final concentration.
Cells were harvested after 4-6 h induction by 30 min of centrifugation (1500 g, 4°C). The wet cell paste was resuspended in 40 ml Tris buffer (20 mM Tris, 0.1% 2-ME, pH 8.0) using an ice/water cooling bath. Lysozyme was added to a concentration of 50 μg/ml and the mixture was stirred for 30 min at room temperature. The solution was sonified for 30 min using a Branson ultrasonifier W-450D (20% power, 50 % pulse cycle) with a macrotip in an ice/water bath. The buffer and soluble proteins were removed by centrifugation at 3000 g (4°C, 30 min). The pellet was washed in 20 ml 1 M urea solution (20 mM Tris, pH 8.0, 0.1%
2-ME). The supernatant was removed by centrifugation (5000 g x 30 min, 25°C). Then the pellet was dissolved in 40 ml buffer (8 M urea, 20 mM Tris, 0.1% 2-ME, pH 8.0). The solution loaded onto a Q-sepharose FF column (Amersham) and the proteins wereeluted from the column by a NaCl gradient (0-100 mM). The yields of OmpG were about 50 mg/L culture.
For expression of the translocator domain of NalP, plasmid pPU320 (residues D776 to F1083 of NalP) was transformed into E. coli BL21 (DE3) (Stratagene) and the NalP protein was purified as described previously [Oomen et al. 2004].
Interaction of OMPs with Skp and LPS
Plasmid pET15_EcOMP85 (purchased from Trenzyme GmbH, Germany) can overproduce YaeT protein with 6xHis-Tag as inclusion body in E. coli. NcoI and BamHI restriction sites were used for cloning YaeT gene into pET15b vector (Novagen) and the yielding plasmid was named as pET15_EcOMP85. After transformation, we purified YaeT following the similar protocol as OmpG purification because both proteins were expressed as inclusion bodies and had close pI, 4.4 of OmpG and 5.1 of YaeT. The yields of YaeT were about 25 mg/L culture.
Purification of R-LPS
E. coli rough mutant F576 was cultivated as described previously [Vinogradov et al.
1999], and its LPS (R2 core type, M≈ 3900 g/mol) was isolatedas reported [Müller-Loennies et al. 1994].
Fluorescence spectroscopy
Fluorescence spectra were recorded as described previously [Bulieris et al. 2003] on a Spex Fluorolog-3 spectrofluorometer with double monochromators in the excitation and emission pathways. The excitation wavelength was 295 nm (unless stated), and the bandwidths of the excitation monochromatorswere 2.5 nm. The bandwidths of the emission monochromators were5 nm. The integration time was 0.05 s, and an increment of 0.5nm was used to scan spectra in the range of 310-380nm. Background intensities of Skp in absence of OMPs were subtracted. These intensities were relatively small since Skp does not contain Trp.
Unless stated otherwise, each experiment was performed three times at same conditions. All the experiments were performed at 25 °C.
Binding of Skp to OMPs monitored by fluorescence spectroscopy
The background spectra of Skp at different concentrations were recorded first in 1 ml of 10mM Glycinebuffer (pH 9.0). After the addition of certain concentration of OMPs, the fluorescence spectra of OMPs were recorded at each Skp concentration. The concentrations of OMPs were 0.37 μM (OmpG), 0.16 μM (YaeT), 0.55 μM (NalP), and 0.83 μM (TMD-OmpA). Binding functions were fitted to the experimental data assuming one class of identical binding sites. In this case, the average concentration of bound Skp, [B], is given by [Van Holde et al. 2006]:
[B] / [tOMP] = n Kass [F] / (1 + Kass [F]) (Eq. 2.1)
Interaction of OMPs with Skp and LPS
where n is the number of binding sites, Kass the association constant, [tOMP] the total concentration of the outer membrane protein, and [F] the concentration of the free ligand.
Substitution of the free Ligand with the total ligand concentration, [L0] = [B] + [F] and some rearrangements lead to:
[B] = ½ { Kass–1 + [L0] + n [tOMP] – ( (Kass–1 + [L0] + n [tOMP])2 – 4 n [tOMP][ L0] )1/2 } (Eq. 2.2) The concentrations of free and bound OMP are then given by [B] and [tOMP]. The fluorescence signal of the OMP in binding experiments is a linear combination of the concentrations of bound and free OMP, since Skp does not contain fluorescent tryptophan.
Skp binding to unfolded OMPs at different pH and NaCl concentration
First, the spectrum of the OMP was recorded in buffer after urea-dilution. Then the spectrum of a 5 fold-excess of Skp was recorded to obtain a spectrum of the background.
After addition of the OMP, the spectrum was recorded again. Similar experiments were performed once with each different pH buffer: 10 mM Citrate (pH 3.0), 10 mM Citrate (pH 4.0), 10 mM Citrate (pH 5.0), 10 mM Citrate (pH 6.0), 10 mM Hepes (pH 7.0), 10 mM Tris (pH 8.0), 10 mM Glycine (pH 9.0), 10 mM Glycine (pH 10.0) and 10 mM CAPS (pH 11.0).
The OMP concentrations were 0.43 μM (OmpA), 0.47 μM (NalP), 0.65 μM (hVDAC1), and 0.20 μM (FomA). In experiments to determine whether the binding of Skp to either OmpA or OmpG depends on the ionic strength, 10 mM Tris (pH 8.0) buffer was used, containing either 0, 0.1, 0.2, 0.5 or 1 M NaCl. The OMP concentrations were 0.43 μM (OmpA) and 0.37 μM (OmpG). The experiments of Skp binding to OmpG were performed at an excitation wavelength of 290 nm.
Dynamic Light Scattering
The hydrodynamic radius and particle mass of Skp in solution were measured by dynamic light scattering using a Dynapro instrument (Wyatt Technology Corp.). Solutions of Skp (2 g/l) in 12 μl of buffer were first filtered through aluminum oxide filters (Whatman) of 0.02 μm pore size. The intensity of the scattered light was measured and the hydrodynamics radius (RH) was evaluated with the program Dynamics, Ver. 6. This radius is related to the theoretical hydrodynamic radius of an ideally spherical particle (RHTH) and the ratio of the frictional coefficients, f/f0 for a hydrated vs non-hydrated sphere:
RH/RHTH=f/f0
Interaction of OMPs with Skp and LPS
The molecular mass of the Skp particles can be calculated from the hydrodynamic radius:
Mr =(4/3) π [(f0 / f) RH] 3/ (VP + HAQ) NA (Eq. 2.3) Vp is the specific volume of the particle (for a protein, an average of 0.73 cm3/g is normally assumed [Cantor et al. 1980], HAQ is the hydration volume of the protein (typically assumed to be 0.35 cm3 / g protein). For a spherical protein, f/f0 = 1.2. For Skp, which resembles a prolate elipsoide, the Perrin factor is 1.02, leading to f/f0 = 1.22.
CD Spectroscopy
Far UV CD spectra were recorded at RT by a Jasco 715 CD spectrometer (Jasco, Tokyo, Japan) using a 0.5 mm cuvette. Three scans were accumulated from 190 to 250 nm with a response time of 8 s, a bandwidth of 1 nm and a scan speed of 50 nm/min. Background spectra without Skp were subtracted. The concentrations of Skp (30 μM) were determined for each sample [Lowry et al. 1951]. The recorded CD spectra were normalized to the mean residue molar ellipticity [Θ](λ), given by
[ ]
Θ ( )λ =100Θ( )λc⋅n⋅l , (Eq. 2.4)
where l is the path length of the cuvette in cm, Θ(λ) is the recorded ellipticity in degrees at wavelength λ, c is the concentration in mol/l, and n the number of amino acid residues of Skp (141).
LPS binding to the complex of OmpA·Skp3
The fluorescence spectrum of the complex of OmpA·Skp3 was recorded after the addition of LPS in 10 mM Tris buffer (pH 8.0). The concentrations of OmpA and Skp are 0.43 μM and 1.3 μM respectively. The LPS concentration ranged from 0 to 13 μM. The mix steps were performed according to buffer→Skp→OmpA→LPS sequence in the experiments.
Fluorescence quenching experiments with acrylamide
Acrylamide quenching experiments were performed in 10 mM Tris buffer (pH 8.0) with 0, 0.1, 0.2, 0.3 and 0.4 M acrylamide. The working concentrations of OmpA, Skp and LPS are 0.85 μM, 4.25 μM and 6 μM, respectively. The data were fitted to the linear Stern-Volmer function: F0/F = 1 + Ksv [Q]. Ksv is the Stern-Volmer constant and [Q] is the concentration of acrylamide. F has been corrected for the inner filter effect for absorption, Fcorr = Fmeas * 101/2*0.55*[Q].
Interaction of OMPs with Skp and LPS