Figure 2.

Quantitative measurement of EhVps20t bound to the membrane

(A) Calibration curve of the average intensity per pixel obtained with different concentrations of OG-DHPE in POPC:POPS (80:20) GUVs equilibrated in protein buffer (25 mM Tris, 150 mM NaCl, pH = 7.4). The symbols represent measurements on different vesicles and the red line is a linear fit (see text for expression).

(B) Quantitative estimate for the coverage of EhVps20t (number of protein molecules per membrane area) for different bulk protein concentration at which POPC:POPS:Chol:PI(3)P (62:10:25:3) GUVs were incubated. In each condition, triplicate incubations were done. Symbols represent the mean value and vertical lines the standard error of the coverage measured on 20 GUVs. The insert shows outward tubulation observed when 1200 nM of EhVps20t was used.

(C) Representative images of the coverage of EhVps20 t at the membrane for two different concentrations (125 nM and 800 nM), both of which are dilute and small compared to conditions of protein crowding. The depicted fractions and squares indicate the average areas occupied by a single molecule of EhVps20t (orange line, in scale with the square size; the thickness of the light blue square border illustrates the error) in the respective conditions. The predicted 3D structure and approximate size of EhVps20t in crystalline state is also given (note that upon binding the protein could unfold and occupy a larger area). Scale bars: 20 μm