Fig. 5

Crucial role of residues T323/M324/R325 in the extension of domain 3a hinge loop. A Position of FRET pair labeled on Syx1 to monitor the relative movement between the linker region and Habc in the crystal structure of Munc18-1/Syx1 (PDB ID: 3C98). A Syx1 S95C/C145S/S171C mutation was generated for stochastic labeling of the FRET pair Alexa-555 and Alexa-647. B Representative histograms of smFRET efficiency of FRET-pairs labeled Syx1 binding Munc18-1 WT or TMR mutant in the absence and presence of MUN domain. Munc18-1/Syx1 was fixed on a coverslip modified with PEG/PEG-NTA-Co²⁺ via the His tag at the C-terminus of Syx1. C TMR mutation impaired Syb2 binding to the Munc18-1/Syx1 complex in the presence of the MUN domain, as detected by GST pull-down. Bands of Munc18-1 were highlighted with red asterisk in Coomassie brilliant blue stained SDS–PAGE. Representative gel displayed is from one of three independent replicates. D Quantification of the integrated densities of the Munc18-1 bands in C. The band density of WT Munc18-1 in the presence of the MUN domain was taken as 1. Data are presented as mean values ± SD, n = 3