Domain swapping oligomerization of thermostable c-type cytochrome in E. coli cells

Knowledge on domain swapping in vitro is increasing, but domain swapping may not occur regularly in vivo, and its information in cells is limited. Herein, we show that domain-swapped oligomers of a thermostable c-type cytochrome, Hydrogenobacter thermophilus cyt c552, are formed in E. coli which expresses cyt c552. The region containing the N-terminal α-helix and heme was domain-swapped between protomers in the dimer formed in E. coli. The amount of cyt c552 oligomers increased in E. coli as the cyt c552 concentration was increased, whereas that of high-order oligomers decreased in the order of decrease in protein stability, indicating that domain swapping decreases in cells when the protein stability decreases. Apo cyt c552 was detected in the cyt c552 oligomer formed in E. coli, but not in that of the A5F/M11V/Y32F/Y41E/I76V mutant. The cyt c552 oligomer containing its apo protein may form at the periplasm, since the apo protein detected by mass measurements did not contain the signal peptide. These results show that domain-swapped cyt c552 oligomers were formed in E. coli, owing to the stability of the transient oligomer containing the apo protein before heme attachment. This is an indication that exceedingly stable proteins may have disadvantages forming domain-swapped oligomers in cells.

Preparation of HT apo cyt c 552 . After reaction of HT cyt c552 with 10 mg/ml Ag2SO4, the solution containing the apo protein was treated with 1 M dithiothreitol and 6 M guanidine hydrochloride. The obtained solution was incubated at 25 °C under dark for 4 h, and subsequently centrifuged (20,000 g, 15 min, 20 °C). The supernatant was dialyzed overnight with 25 mM sodium acetate buffer, pH 5.0, and 25 mM Tris-HCl buffer, pH 8.0, for HT apo cyt c552 without a His-tag and His-tag-attached HT apo cyt c552, respectively. HT apo cyt c552 without a His-tag and His-tagattached HT apo cyt c552 were subsequently purified with HiTrap SP and HisTrap HP columns (GE Healthcare), respectively, using the FPLC system (BioLogic DuoFlow 10, Bio-Rad). The two cysteine residues which were originally bound to the heme formed an intramolecular disulfide bond. Oxidation of the cysteine residues of HT apo cyt c552 was confirmed by Ellman's regent 5 .
X-ray crystallographic analysis. Oxidized dimeric HT cyt c552 without a His-tag was dissolved in 100 mM HEPES buffer, pH 7.0, at a protein concentration of 3.0 mM (heme unit). Droplets prepared by mixing 2 μL of the protein solution with 2 μL reservoir solution were equilibrated. The reservoir solution was 1.6 M sodium citrate buffer, pH 6.5. A crystal was observed in the protein solution after incubation at room temperature for three days.
The crystal was mounted on a cryo-loop without an additional cryoprotectant, and flash-frozen at 100 K in a nitrogen cryo system. The crystal-to-detector distance was 250.0 mm, and the wavelength was 1.0 Å. The oscillation angle was 1°, and the exposure time was 12.0 s per frame. The total number of frames was 180. The diffraction data were processed using the program, HKL2000 6 . The preliminary structure was obtained by a molecular replacement method (MOLREP) 7 using the atomic coordinates of the structure of dimeric HT cyt c555 (PDB code: 3VYM) as a starting model. The structure refinement was performed using the program, REFMAC 8 . The molecular model was manually corrected, and water molecules were picked up in the electron density map using the program, COOT 9 . The data collection and refinement statistics are summarized in Table SI (Supporting Information). The three-dimensional structure of the protomer of the dimer obtained from the E. coli expression system and that obtained by ethanol treatment were compared using the molecular graphics program, PyMOL 10 .
The α-helical content was estimated from the Cotton effect at 222 nm by the equation previously reported 12 .

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The buffer of the sample was exchanged with ultrapure water using an Amicon ultrafiltration tube (Merck Millipore) before mass measurements.
Refolding of HT cyt c 552 . Oxidized His-tag-attached WT and A5F/M11V/Y32F/Y41E/I76V HT holo cyt c552 (0.3 mM) in the unfolded state with and without its apo protein (0.3 mM) were obtained by an addition of guanidine hydrochloride (final concentration, 5.33 M) in 50 mM potassium phosphate buffer, pH 7.0. After the HT cyt c552 solution was passed through the desalting gel column, the solution was incubated at 37 °C for 1h. The fractions were analyzed by Ni affinity chromatography (column: HisTrap HP, GE Healthcare) using the FPLC system (BioLogic DuoFlow 10, Bio-Rad) and MALDI-TOF mass spectroscopy.