Document: The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/276147 doi: bioRxiv preprint Figure S2 . Membrane coverage measurements of N-BAR, Amph-FL, and Amph CTD ∆SH3, and fluorescence correlation spectroscopy (FCS) of Amph CTD ∆SH3. Tethered vesicle composition for N-BAR and Amph-FL: 76 mol% DOPC, 15 mol% DOPS, 5 mol% PtdIns(4,5)P 2 , 2 mol% DP-EG10-biotin, and 2 mol% Oregon Green 488-DHPE. In experiments with Amph CTD ∆SH3, DOPS and PtdIns(4,5)P 2 were replaced by 20 mol% DOGS-NTA-Ni. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/276147 doi: bioRxiv preprint function of protein concentration. Because Amph-FL occupies a greater area on the membrane surface compared to N-BAR (79 versus 16.5 nm 2 per monomer, respectively), Amph-FL reaches a higher coverage than N-BAR at equal number density of membranebound proteins. At 25 nM, Amph-FL reaches approximately 20% membrane coverage, approaching a crowded regime (Snead et al., 2017; Stachowiak et al., 2012) . Markers in (D) and (E) represent mean ± first s.d., n = 3 independent experiments. (F) Membrane coverage estimates for 25 and 100 nM Amph-FL on 30 nm vesicles. At 100 nM Amph-FL, when potent membrane fission occurs, Amph-FL reaches approximately 77% membrane coverage, significantly higher than can be reached by non-assembling proteins. At this coverage, steric pressure from disordered domain crowding is very high, providing a potential explanation for strong membrane fission by Amph-FL. Markers indicate mean of all coverage values, error bars represent 95% confidence interval. 25 nM: n = 2,171 vesicles, 100 nM: n = 1,783 vesicles. (G-I) Representative, normalized FCS traces of (G) Amph CTD ∆SH3, (H) AP180 CTD, and (I) transferrin. Blue dots indicate data, black lines indicate fit (see methods). Average values of diffusion time, t D , ± first s.d. are shown next to each trace, with n = 10, 5, and 3 FCS traces for Amph CTD ∆SH3, AP180 CTD, and transferrin, respectively. The hydrodynamic radius, 6 , of each protein is also shown next to each trace. 6 of Amph CTD ∆SH3 was computed by scaling from the known 6 of AP180 CTD (see methods). Using this same approach yields an 6 of transferrin that is similar to its expected value (Hall et al., 2002) . (J) Relative diffusion times of Amph CTD ∆SH3 in buffer with 10, 150, and 1,000 mM sodium chloride (NaCl), expressed as the proportion of the average diffusion time at 150 mM NaCl. The data indicate a transition from an extended to a more compact state with increasing ionic strength, as expected for charged disordered proteins (Srinivasan et al., 2014) . Diffusion times were corrected for changes in solution viscosity with varying NaCl concentration (Zhang and Han, 1996) . Red dots indicate data, black lines indicate mean. n = 5 FCS traces at each NaCl concentration. (K) Images of tethered vesicles (green, left column) and membrane-bound Amph CTD ∆SH3-Atto 594 (red, right column). Top row is 1 µM, bottom row is 2 µM. Images in each column have equal contrast to show greater protein intensity with increasing concentration. Scale bars: 2 µm. (L) Raw protein intensity as a function of raw vesicle intensity for the 1 µM Amph CTD ∆SH3 dataset. (M) The same 1 µM Amph CTD ∆SH3 dataset after processing, plotted as the area occupied by membrane-bound proteins as a function of vesicle surface area. The slope of a linear fit to the
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