Figure 1

(Color) Probe-partitioning fluorescence resonance energy transfer. Membranes are labeled with trace quantities of fluorescent donor (green) and acceptor (red) probes. In a single-phase membrane, an intermediate FRET signal intensity is observed. In the presence of coexisting phases, however, the probes can partition preferentially between alternative environments, causing their local concentrations to rise or fall. If the probes prefer different phases, they are effectively separated, decreasing FRET. If the probes prefer the same phase, they are effectively clustered and the FRET signal increases.Reuse & Permissions

Figure 2

(Color online) SP-FRET profiles for four different combinations of $\text{donor}\to \text{acceptor}$ probes, carried out in DLPC-DPPC at $20.0°\mathrm{C}$. SP-FRET efficiency ($F_A^{Dex}$, arbitrary units) is plotted vs mole fraction of DPPC. In each profile, a distinct regime of reduced efficiency or enhanced efficiency corresponds to coexisting $L_{\alpha }$ (fluid) and $L_{\beta }$ (solid) phases between ${\chi }_{\mathit{DPPC}}^{\mathit{\text{fluidus}}}=0.235$ and ${\chi }_{\mathit{DPPC}}^{\mathit{\text{solidus}}}=0.785$. Reduced efficiency is evident in (a), (c), and (d), in which donors and acceptors prefer different phases. In (b), enhanced efficiency indicates the preference of both probes for the DPPC-rich $L_{\beta }$ phase. Red lines correspond to best-fit curves in accordance with Eq. (9) and the following partition coefficients: $K^{\mathrm{18:0}-\mathit{DiO}}=7.4\pm 0.2$; $K^{\mathrm{18:2}-\mathit{DiO}}=0.23\pm 0.01$; $K^{\mathrm{18:0}-\mathit{DiI}}=7.5\pm 0.3$; $K^{\mathrm{18:2}-\mathit{DiI}}=0.22\pm 0.01$; $K^{\mathit{DHE}}=0.61\pm 0.02$.Reuse & Permissions

Figure 3

(Color) Resolution of fit degeneracy by coupled fitting of conjugate profiles. Goodness of fit $\left({\log }_{10}{\chi }_{\mathit{red}}^2\right)$ data is plotted over a 10 000-fold range of both donor and acceptor $K^P$ values for two of the SP-FRET profiles from Fig. 2. Left-hand plots illustrate the degeneracy observed when either of the plots is fit in isolation to Eq. (9): The $\mathrm{18:0}\text{−}\mathrm{DiO}\to \mathrm{18:0}\text{−}\mathrm{DiI}$ profile is fit well (i.e., ${\chi }_{\mathit{red}}^2\approx 1$) by a range of complementary $K^D,K^A$ values [blue arc, panel (a)] and the $\mathrm{18:2}\text{−}\mathrm{DiO}\to \mathrm{18:0}\text{−}\mathrm{DiI}$ profile is fit by two symmetric loci in $K^D,K^A$ space [blue spots, panel (c)]. Right-hand plots show that the degeneracy is resolved when the fits are coupled (i.e., using a single merged ${\chi }_{\mathit{red}}^2$ parameter), in which case both SP-FRET curves are best fit by unique $K^D,K^A$ combinations [panels (b) and (d)].Reuse & Permissions

Figure 4

(Color online) An optimization approach yields the same best-fit $K^P$ values. In the experiments shown above, Newton’s method was used for the simultaneous optimization of all four curve fits shown in Fig. 2. Panels (a) and (b) show two independent trials starting from different initialization conditions. The left vertical axis represents $K^P$ values for the two 18:0 probe species, while the right axis shows $K^P$ values for both the 18:2 probes and DHE. All five $K^P$ values converge to the same values reported in the legend for Fig. 2.Reuse & Permissions