Extended Data Fig. 2: NCR247 binds haem and NSR247 lacks haem binding ability.
From: A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria
a, Purified MBP-NCR247 is a reddish colored protein. b, UV-Vis spectra of MBP-NCR247 showing peaks at 362 nm, 418 nm, and 540 nm with a slight shoulder at 580 nm. c, LC-MS spectrum from MBP-NCR247 (top) when compared to heme standard (Bottom). d, Metal content of purified MBP and MBP-NCR247 (measured using ICP-MS analysis) showing the presence of iron and absence of any other metal. Data are presented as mean of three biological replicates ± s.d. e, EPR spectrum of NSR247 with heme (g values =5.56) indicating a high spin ferric heme (spectrum similar to free heme). f, Resonance –Raman spectrum of NSR247 with heme shows prominent ν peaks indicative of a Fe3+, five-coordinate, high spin (5cHS) b-type heme. g and h, Lack of change in expression of iron uptake gene (hmuP) upon treatment with 2 µM (g) or 15 µM (h) NSR247. Data are expressed as starting quantities (SQ) of respective mRNAs normalized to the control gene SMc00128 and are presented as average of three technical replicates ± s.d. ****P < 0.0001 NCR247 treated WT vs Untreated; two-way analysis of variance (ANOVA) with multiple comparisons. i, UV-Vis spectra of NCR247- Ferrous heme complex, after reduction by excess Sodium dithionite in an anaerobic chamber, indicating peaks at 420 nm and 550 nm. j, Representative raw image of association and dissociation steps (before and after diving red line) in an Octet bio-layer interferometry experiment (detailed in methods). Biotinylated heme was used as ligand and NCR247 was used as analyte. In a, b, c, e, f, and i, representative data from three independent experiments is shown.
