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. 2021 Oct 20;297(5):101317. doi: 10.1016/j.jbc.2021.101317

Figure 1.

Figure 1

The B. subtilis GlmM and CdaACDproteins form a complex in vitro.A, FPLC chromatograms and SDS-PAGE gel analysis of the B. subtilis CdaACD and GlmM complex. The B. subtilis proteins CdaACD, GlmM, and the CdaACD/GlmM complex were purified by Ni-affinity chromatography and size-exclusion chromatography. The FPLC elution profiles, recorded at a wavelength of 280 nm, are shown for CdaACD (blue), GlmM (cyan) and the CdaACD/GlmM complex (pink). Proteins from the peak fractions of the CdaACD-GlmM complex were separated on a 12% SDS PAGE gel and proteins visualized by Coomassie staining (shown in the insert). B, FPLC chromatograms and SDS-PAGE gel analysis of the B. subtilis CdaACD and GlmMF369 complex. Same as in (A) but using the C-terminally truncated GlmMF369 variant in place of the full-length GlmM protein. The experiments were performed in triplicates and representative chromatograms and gel images are shown. C, MST experiment to determine the binding affinity between CdaACD and GlmM. Increasing concentrations of GlmM were mixed with fluorescently labeled CdaACD resulted in a gradual change in thermophoresis and fluorescence readings. The normalized fluorescence values multiplied by a factor of 1000 Fnorm (1/1000) were plotted using the NT Analysis Software (NanoTemper Technologies GmbH) to yield the binding curve. The data points are the average values and standard deviations from five independent MST runs. The Kd was determined from these data using the NT Analysis Software (NanoTemper Technologies GmbH).