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, 9 (1), 10566

Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye

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Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye

Junko Ishikawa et al. Sci Rep.

Abstract

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Electropherograms of standard metalloproteins holo- and apo-transferrin (Tf) by Native-PAGE (a), by SDS-PAGE (b) and by MICS-BN-PAGE (c); ceruloplasmin (Cp) by MICS-BN-PAGE (d); and superoxide dismutase (SOD) by MICS-BN-PAGE (e).
Figure 2
Figure 2
Concept of HAC-2D MICS-BN-PAGE/metal detection PAGE methodology. Panel I: 1D MICS-BN-PAGE in two lanes. Lane A was subjected to the in-gel holo/apo conversion procedure (as in Panel II-1), and then Lane A was subjected to 2D MICS-BN-PAGE after holo/apo conversion (as in Panel III). Lane B was subjected to Cu2+ and Fe3+ detection PAGE (as in Panel II-2). The 2D electropherogram is for a mixture of metalloproteins (holo-Tf, apo-Tf, holo-Cp and holo-SOD), in which holo-metalloproteins in the original sample were isolated as spots off the diagonal line (the dotted yellow line in Panel III). Full-size versions of electropherograms represented in Fig. 2 are depicted in Fig. S2.
Figure 3
Figure 3
HAC-2D MICS-BN-PAGE employing silver staining (a), with Cu2+ detection PAGE (b), of a soluble fraction obtained from R. gelatinosus cells grown in the presence of 1.2 mM Cu2+ (total protein: 31.5 µg). The off-diagonal spot indicated by the asterisk in (a) was subjected to MALDI-TOF MS (Supplementary Fig. S7), identifying the sequences shown in red font as part of the CopI mature sequence (c). Full-size versions of electropherograms represented in Fig. 3 are depicted in Supplementary Fig. S8.
Figure 4
Figure 4
Typical electropherograms from CZE experiments for: 10 µM holo- or apo-Tf without CBB G-250 (upper green trace); and mixtures containing 5 mM CBB G-250 dye with 10 µM holo-Tf (middle blue trace) or with 10 µM apo-Tf (lower red trace).
Figure 5
Figure 5
Results from AutoDock Vina calculations: the Fe-binding site structures for holo-Tf (left) and apo-Tf (right) complexes with CBB-G 250 dye (a); the dye-binding number cumulative frequency distribution as a function of binding energy for holo-Tf (blue) and apo-Tf (pink) (b). Blue, red and yellow atoms in (a) represent CBB G-250, Fe3+ and amino acid residues bound with Fe3+, respectively.

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