2.1. Cloning and expression
The gene encoding PorB was amplified from a clinical isolate of N. meningitidis
(Kilic et al.
; a generous gift from Dr YiWei Tang, Department of Infectious Disease, Vanderbilt University Medical Center) using the previously described sense (5′-GGG GTA GAT CTG CAG GTT ACC TTG TAC GGT ACA ATT AAA GCA GGC GT) and antisense (5′-GGG GGG GTG ACC CTC GAG TTA GAA TTT GTG ACG CAG ACC AAC) primers (Feavers et al.
). The PCR-amplified fragment was digested with Bgl
I and then cloned into the Bam
I sites of the pET21b expression vector without any affinity tags. Recombinant PorB expression was performed using modified Escherichia coli
BL21 (DE3) cells (Locher & Rosenbusch, 1997
). Cells were grown in LB medium at 303 K with 100 µg ml−1
ampicillin until an OD600
of 0.5 was reached. PorB expression was induced by the addition of isopropyl β-d
-1-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM
. The cells were incubated with shaking for 4 h at 310 K and then harvested by centrifugation at 5000g
for 20 min at 277 K. Using this protocol, PorB was expressed into inclusion bodies (Qi et al.
). Cells expressing seleno-l
-methionine-incorporated PorB were grown in minimal medium supplemented with 25 mg l−1
-methionine (SeMet) by inhibition of the methionine-biosynthesis pathway (van Duyne et al.
2.2. Protein purification
Purification of N. meningitidis
PorB was modified from a previously described protocol (Qi et al.
; Tanabe & Iverson, 2009
). Cell pellets were resuspended in breaking buffer (10 mM
Tris–HCl, 5 mM
EDTA pH 8.0) supplemented with 1 µg ml−1
DNaseI and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) and were lysed by sonication on ice (60 Sonic Dismembrator, Fisher Scientific). The insoluble material was isolated by centrifugation at 30 000g
for 30 min in an SS-34 rotor (Sorvall). The pellet was resuspended in TSE buffer (20 mM
Tris–HCl pH 7.5, 200 mM
NaCl, 1 mM
EDTA) supplemented with 2% Triton X-100. The suspension was stirred for 1 h and insoluble material was sedimented by centrifugation at 30 000g
for 20 min. The pellet was washed three times with TSE buffer supplemented with 2% Triton X-100 and then with TSE buffer alone in order to remove the detergent.
Approximately 1 g of inclusion bodies (from 400 ml LB culture) was resuspended in 1 ml denaturing buffer (50 mM Tris–HCl pH 7.5, 200 mM NaCl, 7.2 M urea) and sonicated for 1 min in a bath sonicator; it was then quickly diluted with 2 ml detergent buffer [50 mM Tris–HCl pH 7.5, 200 mM NaCl, 10% lauryldimethylamine N-oxide (LDAO)] and sonicated for 1 min in a bath sonicator. After 10 min incubation, the insoluble material was sedimented by centrifugation at 20 000g for 20 min. The clarified soluble material was passed through a 0.22 µm filter and loaded onto a Hi-Load Superdex 200 16/60 size-exclusion column equilibrated with TSE buffer supplemented with 0.15% LDAO. The elution time was consistent with a trimer (Fig. 1
a). For SeMet PorB, all buffers were supplemented with 5 mM 2-mercaptoethanol.
Figure 1 Purification of N. meningitidis PorB. (a) Size-exclusion chromatogram of PorB on a Hi-Load Superdex 200 16/60 size-exclusion column (120 ml bed volume) following refolding from inclusion bodies. Two peaks are observed. The first peak elutes at (more ...)
Prior to crystallization, the purified protein was concentrated to 15 mg ml−1 in crystallization setup (CS) buffer (20 mM Tris–HCl pH 7.5, 200 mM NaCl) supplemented with one of several types of zwitterionic detergents [0.1% LDAO, 0.01% tetradecyl-N,N′-dimethylglycine (TDDG), 0.02% Fos-choline 14, 0.002% Fos-choline 16, 0.2% LAPAO (3-laurylamido-N,N′-dimethylpropyl aminoxide), 0.3% Anzergent 3-12 or 0.03% Anzergent 3-14]. The sample purity was assessed by SDS–PAGE of protein samples suspended in SDS sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 5% glycerol, 1% 2-mercaptoethanol and 0.004% bromophenol blue) without heat treatment (Fig. 1
b). The identity of PorB was independently confirmed by protein excision, in-gel digestion with trypsin protease and subsequent MALDI–TOF/TOF and ESI–LC/MS/MS mass spectrometry at the Vanderbilt University Proteomics core facility, giving a combined coverage of 89.5% of the predicted protein sequence. The β-sheet percentage of PorB at 0.7 mg ml−1 was confirmed by circular-dichroism (CD) spectroscopy (Fig. 1
c) at 298 K using a Jasco J-810 CD spectropolarimeter. The solution was in a 1 mm path-length cell and the spectra were recorded with 20 nm min−1 scanning time. The protein concentration was estimated using the BCA protein assay (Pierce).
Initial sparse-matrix crystallization screening of 15 mg ml−1 PorB sampled 1440 chemically distinct crystallization conditions using a Mosquito crystallization robot (TTP LabTech). Both sitting and hanging drops comprised of 200 nl protein solution plus 200 nl reservoir solution were equilibrated against 100 µl reservoir solution at either 277 or 291 K. Following the identification of preliminary crystallization leads, crystals were optimized manually using the hanging-drop vapor-diffusion method with 1 µl protein solution plus 1 µl reservoir solution incubated over 1 ml reservoir solution.
Crystal form 1 (C222; Fig. 2
a) was grown by mixing 1.0 µl protein solution (CS buffer plus 0.1% LDAO, with protein at 15 mg ml−1) with an equal volume of reservoir solution containing 50 mM Tris–HCl pH 8.5, 13%(w/v) PEG 1500, 0.03%(w/v) Anzergent 3-10 and 3%(w/v) DMSO. Crystals grew to approximately 0.2 × 0.2 × 0.1 mm at 291 K and formed in between 3 and 5 d. Prior to data collection, the crystals were quickly dragged through crystallization solution supplemented with 30%(w/v) PEG 1500 and 15%(w/v) glycerol for cryoprotection and were flash-cooled in liquid nitrogen.
Figure 2 Crystals of PorB from N. meningitidis. Three crystal forms of N. meningitidis PorB have been grown using the hanging-drop vapor-diffusion method. (a) Crystals of PorB in space group C222 were grown using PEG 1500 as a precipitant. (b) PorB crystals in (more ...)
Crystal form 2 (R32; Fig. 2
b) and crystal form 3 (P63; Fig. 2
c) were grown by mixing 1.0 µl protein solution [CS buffer plus 0.01%(w/v) TDDG, with protein at 15 mg ml−1] with an equal volume of reservoir solution containing 100 mM MES buffer pH 6.0–6.5, 50 mM CsCl, 28–32%(w/v) Jeffamine M-600. The R32 crystals preferentially formed from native PorB and grew to maximum dimensions of approximately 0.10 × 0.10 × 0.02 mm in 2–3 weeks at 291 K. Prior to data collection, the crystals were quickly dragged through a crystallization solution with the Jeffamine M-600 concentration increased to 35–40%(w/v) for cryoprotection, followed by flash-cooling in liquid nitrogen. The P63 crystal form used the same crystallization conditions as the R32 crystal form but preferentially grew from SeMet-incorporated protein. The crystals grew to maximum dimensions of approximately 0.05 × 0.05 × 0.1 mm and formed in 2–3 months.
2.4. Data collection and processing
Crystal quality was assessed by diffraction on Stanford Synchrotron Radiation Laboratory (SSRL) beamlines 9-2 and 11-1, Advanced Light Source (ALS) beamlines 8.2.2 and 8.3.1, Cornell High Energy Synchrotron Source (CHESS) beamlines A1 and F2 and the Advanced Photon Source (APS) Industrial Macromolecular Crystallography Association (IMCA-CAT; ID-17), Southeast Regional Collaborative Access Team (SER-CAT; ID-22) and Life Sciences Collaborative Access Team (LS-CAT; ID-21-D/F/G) beamlines. Iterative rounds of detergent and additive screening improved the diffraction quality of the crystals as assessed by diffraction-based feedback using synchrotron radiation. Even using this procedure, the best crystals of crystal form 1 (C222) diffracted to a maximum resolution of 5 Å after careful optimization of the crystallization conditions. As a result, this crystal form was abandoned for structure determination.
Following optimization, crystal form 2 (R32) diffracted isotropically to 2.9 Å resolution (Fig. 3
a) and a complete native data set was collected at 100 K on SSRL beamline 11-1 using a MAR 325 CCD detector. The exposure time was 15 s for each frame and used an oscillation width of 1° per image.
Figure 3 Typical X-ray diffraction patterns of the R32 and P63 crystal forms. (a) Diffraction image from the R32 crystal form collected on SSRL beamline 11-1. The oscillation angle was 1°. Circles superimposed onto the diffraction pattern correspond to (more ...)
Data for both native and SeMet-incorporated PorB in crystal form 3 (P
) were collected using a wavelength of 0.978 Å at the APS LS-CAT ID21-G on a MAR 225 CCD detector. A data set consisting of 120 frames was collected with a rotation angle of 90° and an exposure time of 5 s per frame. All data were processed and scaled using the HKL
-2000 program package (Otwinowski & Minor, 1997