Extended Data Fig. 2: Dynamics of teixobactin in the complex and pyrene excimer fluorescence confirm Lipid II oligomerization upon binding of teixobactin.
From: Teixobactin kills bacteria by a two-pronged attack on the cell envelope
a) Site-resolved 15N T1rho dynamics of Lipid II-bound teixobactin in membranes acquired at 60 kHz (MAS). The overall dynamics of the molecule is very slow, in agreement with the immobilization of teixobactin in large oligomeric complexes. The error bars indicate the standard error of the fit. Data represented as mean +/− SD. Source data are provided. Monomeric pyrene fluorescence emission maxima occur at 378, 398, and 417 nm. Pyrene molecules exhibit a unique fluorescence at 490 nm only when two pyrene molecules come within 10-20 Å of each other, forming an excited dimer state known as the excimer state. Source data are provided. b) Fluorescence spectra of pyrene-labelled Lipid II in LUVs for increasing concentrations of teixobactin. It can be readily seen that with the increase in teixobactin concentration there is a relative decrease in the monomer signal followed by a respective increase in the excimer signal. Each spectrum is acquired immediately after the addition of teixobactin and averaged over 50 s, demonstrating that the oligomerization event is almost instantaneous. c) The insert shows the excimer region of the plot. d) Quantification of the excimer over monomer ratio as a function of the teixobactin concentration. e) Fluorescence microscopy shows oligomerization and membrane perturbations. A 3D render of the z-stack of DOPC GUVs doped with Atto-labelled Lipid II and treated with teixobactin shows Lipid II oligomerization. f) A 2D plane of the same GUV shown in e) revealed membrane perturbations induced by the oligomers. ii) Transmission image of the GUV. g) Control GUV: i) fluorescent ii) Transmission image; h) i) Confocal microscopy images of GUVs taken from biological triplicates ii) The transmission images. All scale bars represent 5 μm.
