Domain-swapped cytochrome cb 562 dimer and its nanocage encapsulating a Zn–SO4 cluster in the internal cavity† †Electronic supplementary information (ESI) available: Experimental procedure, size exclusion chromatogram, DSC thermogram, protein structure and statistics of data collection. See DOI: 10.

Three domain-swapped cytochrome cb 562 dimers formed a unique cage structure with a Zn–SO4 cluster inside the cavity.


Supplementary figures and table Fig. S1
Size exclusion chromatograms of oxidized E. coli cyt b562 and cyt cb562.

Fig. S2
Size exclusion chromatograms of the solution containing dimeric cyt b562 and dimeric cyt cb562.

Fig. S4
Overlapped view of the protein and active site structures of monomeric and dimeric cyt cb562.

Fig. S5
Fo − Fc omit map of the Zn 2+ ions in the internal cavity of the cage structure of dimeric cyt cb562.

Fig. S6
Coordination structures of Zn 2+ and SO4 2ions in the internal cavity of the cage structure of dimeric cyt cb562.

Fig. S7
Schematic views of the hydrogen bonds at the hinge loop for the protomers of dimeric cyt cb562 and monomeric cyt b562.

Fig. S8
Comparison of Zn binding sites containing Ala1 and Asp39 between the domain-swapped cyt cb562 dimer cage and cyt cb562 surface mutant cage.

Table S1
Crystallographic statistics of data collection and structure refinement of dimeric cyt cb562.

Supplementary references p. S15
Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2015

Experimental section
Plasmids of E. coli cyt b 562 and cyt cb 562 The E. coli cyt b562 gene was synthesized (Life Technologies Japan, Tokyo) and sub-cloned into the NdeI-BamHI site of the pET29b plasmid. Conversion of cyt b562 to cyt cb562 (R98C/Y101C cyt b562 mutant) was based on reported method. 1   Cyt cb562 was oxidized by the same procedures as cyt b562. The purity of cyt cb562 was confirmed by the ratio of the absorbance at 415 nm to that at 280 nm (Abs415/Abs280 > 8). Molar extinction coefficients of oxidized monomeric and dimeric cyt cb562 were determined by the hemochrome method as ε415 = 136 ± 1 and ε415 = 133 ± 1 mM -1 cm -1 , respectively. 4 The concentration of cyt cb562 was adjusted by the intensity of its Soret band.

Preparation of dimeric cyt b 562 and dimeric cyt cb 562
Oxidized dimeric E. coli cyt b562 and dimeric cyt cb562 were prepared by an addition of 75% h to remove the unstable dimers. After the incubation, dimeric cyt cb562 was purified using the same column and FPLC system described above.

Size exclusion chromatography analysis
The amount of oxidized dimeric cyt b562 and cyt cb562 in each solution was analyzed by size exclusion chromatography with a Superdex 75 10/300 GL gel column (GE Healthcare) using buffer, pH 7.0; temperature, 4 °C). The elution curves were fitted with a multi-peak Gaussian fitting procedure (Origin 8, OriginLab Corporation). The percentages of the monomer and dimer were obtained by dividing the area of the peak by the total area of the peaks in the elution curve. Diffraction data were collected at the BL38B1 beamline at SPring-8, Japan, using a Quantum315 detector (ADSC). 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 220 mm, and the wavelength was 1.0000 Å. The oscillation angle was 0.5°, and the exposure time was 4 s per frame. The total number of frames was 270. The diffraction data were processed using the program, HKL2000. 7 The preliminary structure was obtained by a molecular replacement method (MOLREP) using the atomic coordinates of the structure of an     The Zn 2+ ions are shown as green, cyan, magenta, orange, red, yellow, and blue spheres. The amino nitrogen and carbonyl oxygen atoms of Ala1, the side chain oxygen atoms of Asp12