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Figure 3
Interaction studies using size-exclusion chromatography (SEC) and Ni–NTA pull-down. (a) (i) SEC profiles of PhCheF (39.7 kDa; gray dashed line), PhCheY (13.1 kDa; black dashed line) and a 1:1 stoichiometric mixture of proteins (black line). (ii) Calibration with ribonuclease (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), albumin (66.2 kDa) and aldolase (158 kDa). The peaks for PhCheY and PhCheF correspond to apparent molecular weights of 15 kDa and 41 and 127 kDa, respectively (calculated molecular weights of 13.1 and 39.7 kDa, respectively). The higher oligomeric species seen for PhCheF corresponds in its apparent molecular weight to a dimeric or trimeric complex. A mixture of PhCheF and PhCheY does not form complexes under the conditions of the experiment. (b) Coomassie-stained SDS–PAGE gel (NuPAGE, 4–12%, Invitrogen, USA) of cross-linking studies with glutaraldehyde. Samples 1–4 reflect different incubation times before quenching (1, 2, 5 and 10 min). For PhCheF, a pronounced dimeric species appears under moderate cross-linking conditions, while PhCheY seems to successively polymerize from monomers. The SDS–PAGE image was modified in contrast. (c) Coomassie-stained SDS–PAGE gel (NuPAGE, 4–12%, Invitrogen, USA) of the Ni–NTA pull-down assay with PhCheF and His-tagged PhCheY. Gels (i) and (ii) show controls with PhCheY and PhCheF, respectively; gels (iii) and (iv) show pull-down with nontreated (iii) and BeF3-treated (iv) PhCheY. PhCheF elutes with PhCheY in (iii) and (iv), suggesting specific interaction. Lane M, marker (PageRuler, unstained protein ladder; Thermo Scientific, USA); lane 1, control (samples incubated without Ni beads); lane 2, control (supernatant after incubation with Ni beads); lanes W1, W2 and W3, wash fractions; lanes E1, E2 and E3, elution fractions. The SDS–PAGE images were modified in contrast.

ISSN: 2053-230X
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