Oligomeric state of YukE.

A. Purified YukE-His₆ from the affinity chromatography step was loaded into a size exclusion column (see methods) and the eluted material was continuously monitored by taking absorbance measurements at 280 nm (A_{280 nm}). The elution volume (ml) of the YukE-His₆ peak (solid curve) and the derived Stokes radius (R_s) and relative mass (M_r) are indicated. Note that the mass was estimated assuming that YukE-His₆ has the same hydrodynamic properties of the globular polypeptides composing the protein standard (dashed curve). The column void volume (V₀), the elution volume of standard proteins and their corresponding masses are also indicated. B. SDS-PAGE and Coomassie blue staining of YukE-His₆ from the peak fraction of the size exclusion chromatography. C. Modeling of the YukE subunit 3D structure by Phyre² (97% YukE sequence coverage, 99.8% confidence, template = GBS1074). D,E. In vitro cross-linking of YukE. Two concentration sets of purified YukE-His₆, from 84 to 20 µM (D) and from 10.3 to 0.4 µM (E) were incubated in the presence or absence of the cross-linking agent BS³. After SDS-PAGE separation (D, 2 µg protein per lane; E, 50 ng protein per lane), the cross-linking products were revealed by Coomassie blue staining (D) or by immunodetection with anti-YukE-His₆ antibodies (E). F. In vivo YukE cross-linking. YukE present in a concentrated culture supernatant of strain W654 (2.5 µg total protein, see methods for details) was cross-linked in presence of 1 or 5 mM BS³ and the cross-linked products detected with anti-YukE-His₆ as in panel E.