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  • Hazel Teague posted an update 1 year, 5 months ago

    ( 12). FCCS was corrected for cross talk (5%) and for labeling degree (100% for BaxG, 80% for BaxR, 90% for Bcl-xLG, and 70% for Bcl-xLR). The percentage of GUV filling was calculated as equation(1) [(Ftin?F0)(Ftout?F0)]��100,where Erlotinib nmr Ftin and Ftout are the average fluorescence intensities inside and outside a GUV, and F0 is the background fluorescence at time t. We arbitrarily set the threshold for a nonpermeabilized GUV to <15%. Several hundreds of GUVs were analyzed per experiment. To quantify protein binding to GUV membranes, the fluorescence intensity at the vesicle rim (Frim) was calculated with Image J using the plug-in radial profile plot. For Fig.?1, intensity was plotted as Frim/Fback, where Fback is the background intensity outside the GUVs. In the kinetics experiments, images were recorded every 20?s and changes in the fluorescence intensity inside GUVs were analyzed over time as equation(2) FtN=[(Ftin?F0)(Ftout?F0)],where FtN is the normalized fluorescence intensity at time t. To calculate the initial A0 and relaxed Arelax permeabilized area of individual GUVs, as well as the relaxation time ��relax, we used a multiexponential fitting described by Eq. 7: equation(3) F(t)inN=1?e?t��flux(t),where the influx rate, ��flux, decreases with time (the initial pore size relaxes to a smaller structure) according to equation(4) ��flux(t)=VA(t)��Dm,where V is the vesicle volume, D the dye diffusion coefficient, m the membrane thickness, and A the total permeabilized area, which varies this website with time according to equation(5) A(t)=Arelax+(A0?Arelax)��e?t��relax.Membrane thickness was assumed to be 4.5?nm. The diffusion coefficient of cyt c-al488 was 196 ��m2/s (196 �� 27 ��m2/s) as calculated by fluorescence correlation spectroscopy (FCS). To compare the permeabilizing activity of Bax and Bcl-xL, it?is essential to analyze their membrane association. We directly visualized and quantified protein binding to membranes by the increase in fluorescence intensity on the rim of individual GUVs (Fig.?1, A and B, and Fig.?S3). cBid enables Bax, as well as Bcl-xL, insertion into the MOM and into model membranes ( 12?and?21). In agreement with its role Metformin as a receptor ( 7?and?12), we observed that membrane binding of cBid (labeled with Al633 or Al488 (cBidR or G)) depends on cardiolipin (CL) and increases with CL concentration ( Fig.?S2). Interestingly, cBidR or G binding to a lipid mixture that mimics the MOM (see Materials and Methods) and contains 6% CL (mol/mol) was too weak to be detected using this method ( Fig.?1D). This weak binding is in line with previous results using different methods ( 23). In the case of Bax (labeled with Atto488 (BaxG) or Atto 655 (BaxR)), its association depended on the presence of both cBid and CL (Fig.?1, C and D, and Fig.?S3). Using the mixture mimicking the MOM, BaxG or R bound to GUVs, but it was close to the detection limit, and with 20% CL the contrast was still weak.