Divide and Conquer: A Tailored Solid‐state NMR Approach to Study Large Membrane Protein Complexes

Abstract Membrane proteins are known to exert many essential biological functions by forming complexes in cell membranes. An example refers to the β‐barrel assembly machinery (BAM), a 200 kDa pentameric complex containing BAM proteins A–E that catalyzes the essential process of protein insertion into the outer membrane of gram‐negative bacteria. While progress has been made in capturing three‐dimensional structural snapshots of the BAM complex, the role of the lipoprotein BamC in the complex assembly in functional lipid bilayers has remained unclear. We have devised a component‐selective preparation scheme to directly study BamC as part of the entire BAM complex in lipid bilayers. Combination with proton‐detected solid‐state NMR methods allowed us to probe the structure, dynamics, and supramolecular topology of full‐length BamC embedded in the entire complex in lipid bilayers. Our approach may help decipher how individual proteins contribute to the dynamic formation and functioning of membrane protein complexes in membranes.


BamABCDE sample preparation Cloning
We modified the pJH114 plasmid in order to obtain independent plasmids for the expression of the BamABDE complex and BamC protein as described below.

pBAD_BamABCDE plasmid
The promoter region of the pJH114 plasmid was exchanged with that of the pBAD plasmid. Amplification of the promotor region of the pBAD vector (3188-294) included the introduction of restriction sites ApaI (3188) and NdeI (294) using the primers FW_pBAD (5'-ATA ATA GGG GCC CTT ATG ACA ACT TGA CGG CTA CAT C-3') and RV_pBAD (5'-ATA ATA CCA TAT GAA AAC GGG TAT GGA GAA ACA GTA-3'). The resulting PCR reaction was treated with DpnI at 37°C for an hour prior to purification with Agencourt AMPure XP (Beckman Coulter). In order to introduce the new promotor, pJH114 must be treated with NdeI and ApaI (this removes the LacQ and Trc promoter region) and then ligate the PCR product in the vector. The vector was gel purified, briefly, 2 μL loading buffer was added to the 15 μL restriction reaction and run on a 0.8 % agarose gel for 2 h. The band corresponding to the linearized plasmid was excised, and DNA was extracted with a Qiagen gel extraction kit. The pBAD_BamABCDE vector was obtained by ligation of the obtained pBAD promotor gene into the NdeI and ApaI pre-treated pJH114 vector. After 5 h incubation at 37°C, 1 μl CIAP, T4 ligase was added and incubated for a further 1 h before 10 min inactivation at 65°C for 10 min.

pBAD_BamABDE plasmid
The pBAD_BamABDE plasmid was obtained by removal of the bamC gene from the pBAD_BamABCDE plasmid. NarI restriction sites were introduced by mutagenesis at the N-and C-terminus of the bamC gene within the plasmid pBAD_BamABCDE as a template and primers FW_C_Nter (5'-ACT CTA TTA CAC GTT AAT CGG CGC CTA GGG AGA TTT GAT GGC TT-3') and RV_C_Cter (5'-GCT GCG TTT AGC AAG TAA GGC GCC TGA GGA AAG TCA AAA CGT-3'). The resulting PCR reaction was treated with DpnI at 37°C for an hour prior to treatment with NarI to remove the bamC gene. The resulting linear vector was purified by gel extraction. Briefly, 2 μL loading buffer was added to the 15 μL restriction reaction and run on a 0.8% agarose gel for 2 h. The band corresponding to the linearized plasmid was excised and DNA was extracted with a Qiagen gel extraction kit. The pBAD_BamABDE vector was obtained by ligation of the obtained NarI pre-treated pBAD_BamABCDE vector. After 5h incubation at 37°C, 1μl CIAP, T4 ligase was added and incubated for a further 1 h before 10 min inactivation at 65°C for 10 min.
The resulting mixtures were transformed into E. coli DH5α cells for plasmid preparation and all DNA sequences were confirmed by sequencing.

a. Unlabeled BamABDE
For the BamABDE complex and BamC, E. coli Lemo21(DE3) cells were transformed with the pBAD_BamABDE plasmid and plated on LB/agar plates supplemented with chloramphenicol and ampicillin, as well as 0.4% glucose. Minimum M9 medium cultures with glycerol (5g/L) instead of glucose were inoculated and grown at 37°C to an optical density of 0.8 after which they were induced with 2 g/L arabinose and incubated for 3.5 h at 37°C.

b. Fractional deuteration of 13 C, 15 N labeled BamC
For expression of the BamC protein, E. coli Lemo21(DE3) cells were transformed with the pCDF_BamC plasmid. Deuterated minimum M9 medium (99% D2O) cultures supplemented with 13 C glucose and 15 N NH4Cl were inoculated and grown at 37°C to an optical density of ~0.7, after which they were induced with 0.5 mM IPTG. Cultures were grown for an additional 7h at 37°C. Cultures of BamABDE and BamC overexpressing cells were combined in a 4:1L ratio to ensure saturation of BamABDE complexes and harvested jointly (4 000xg, 4°C, 20 min). The resulting cell pellet was washed with PBS and frozen at -20°C.

Complex purification and reconstitution into liposomes
The cell pellet was washed with 50 mM Sodium phosphate pH 8.0, 150 mM NaCl, 10 mM imidazole, 1 mM β-mercaptoethanol, proteinase inhibitors, and lysozyme. Lysis was achieved by multiple passes through a French press (3x 10 Kpsi) followed by centrifugation at 4 000xg for 15 min at 4°C. The supernatant was ultracentrifuged at 80 000xg for 45 min at 4°C. The resulting membrane pellet was dissolved in 50 mL 50 mM Sodium phosphate pH 8.0, 150 mM NaCl, 10 mM imidazole, 1% n-Dodecyl β-D-maltoside (DDM, Avanti), proteinase inhibitors and incubated for 3h at 4°C. The solubilized membranes were ultracentrifuged for 45 min at 80 000xg for at 4°C and subsequently filtered before addition to Ni-NTA agarose beads (Qiagen) and incubated overnight at 4°C. The Ni-NTA beads were washed with 20 column volumes of 50 mM Sodium phosphate pH 8.0, 150 mM NaCl, 25 mM imidazole, 0.03% DDM. Elution was achieved with 4 column volumes of 50 M Sodium phosphate pH 8.0, 150 mM NaCl, 0.3 M imidazole and 0.03% DDM. The sample was concentrated with an Amicon ultra-15 centrifugal unit (50 kDa cut-off, Millipore Sigma) before application to a pre-equilibrated Superdex 200 16/60 (GE Life Sciences), with 50 mM Sodium phosphate, 0.1 M NaCl, 0.5 mM ß-mercaptoethanol, 0.03% DDM and run on this column at 1 ml/min. The resulting fractions were pooled, and concentration was determined with the measurement of the absorbance at 280nm on a nanodrop. The BamABCDE complex was reconstituted with DLPC lipids at a 100:1 mol/mol lipid-to-protein ratio via dialysis against 20 mM Sodium phosphate, pH 7.0 at 4⁰C for approximately a week with multiple changes to the dialysis buffer. Liposomes were harvested at 4 000xg at 4°C for 30 min. If the sample was treated with a paramagnetic agent, the liposomes were additionally washed with 10 mM gadodiamide (Omniscan, GE Healthcare). The resulting liposome pellet was packed into a 1.3 mm rotor (Bruker Biospin) prior to the measurements.

Solid-State NMR experiments
The proton-detected NMR experiments of [ 2 H, 13 C, 15 N] labeled BamABCDE samples were carried out at 55kHz on a wide bore 800MHz spectrometer equipped with a 1.3 mm HXY MAS probe (Bruker Biospin). The actual sample temperature was 303K at 55kHz spinning, calibrated by an external DSS standard sample. The 2D NH, 3D CaNH, and CoNH experiments were implemented as described previously [1] . The parameters for each transfer step and acquisition are listed in Tables S1 and S2, respectively. Each 3D spectrum for BamABCDE was recorded in approximately one week of measurement time by adding data sets of 1-day length. During the measurement periods, the magnetic field drifting was monitored by the H2O signal positions and compensated later. The PRE effects are calculated as the peaks intensities ratios on the 2D NH spectra of Gd3+ doped and reference sample and normalized by the number of scans of each spectrum. Figures   Fig. S1. size-exclusion chromatography of the BamABCDE complex. Representative size exclusion chromatography curve obtained for the BamABCDE complex in detergent on an S200 16/60 PG column. The main peak (50-70 mL) was utilized for subsequent sample preparation, displaying the homogeneity of a correctly formed and well-behaving complex, eluting at a volume identical to the BamABCDE complex (~52 mL).

Fig. S2.
BamC chemical-shift analysis. The residue-specific difference in chemical shifts obtained in the current solid-state NMR study and previous results. A) Chemical-shift values for the two folded domains were taken from solution-state NMR assignments (BMRB 16035), while the Nterminal chemical shifts are predicted from the BamABCDE X-ray structure (PDB 5D0Q). B) The chemical shifts difference between solid-state NMR results and the prediction values based on the transmembrane model from the reference [2] . Fig. S3. Cross-sections of 3D 1 H-detected spectra of the BAMABCDE complex. 2D planes of 3D CaNH and 3D CoNH are shown at the most crowded region, as indicated by the red dash line on 2D NH. The assigned peaks were labeled.