Fig. 1. Fluorescence emission spectrum of AnxA2^acryl. AnxA2^acryl (solid line); AnxA2^acryl at pCa3 (dashed line); AnxA2^acryl at pCa3, with LUV (PC/PS 75/25) (L/P = 100) (dotted line). Total protein concentration: 0.5 μM in a 120 μL microcuvette. Excitation wavelength 390 nm (slit width 10 nm), emission slit width: 5 nm.

Fig. 2. Stern–Volmer plots of quenching of acrylodan by iodide. A, AnxA2^acryl: AnxA2^acryl in buffer (●): AnxA2^acryl at pCa3 (○); AnxA2^acryl LUV L/P = 100 at pCa3 (■). B, (AnxA2^acryl-p11)₂: (AnxA2^acryl-p11)₂ in buffer (●): (AnxA2^acryl-p11)₂ at pCa3 (○);(AnxA2^acryl-p11)₂ LUV L/P = 100 at pCa3 (■).

Fig. 3. Time-resolved fluorescence intensity decay of AnxA2^acryl and (AnxA2^acryl-p11)₂. Instrumental response function (curve 1); experimental decays of: AnxA2^acryl (curve 2); AnxA2^acryl pCa 2.7 (curve 3); AnxA2^acryl bound to LUV (PC/PS 75/25) L/P = 100 at pCa 2.7 (curve 4); (AnxA2^acryl-p11)₂ (curve 5). Excitation wavelength: 392 nm, emission wavelength: 505 nm for curves 2, 3 and 480 nm for curves 4, 5. Experimental conditions are shown in the legend to Table 2.

Fig. 4. MEM reconstructed excited state lifetime distribution of AnxA2^acryl and (AnxA2^acryl-p11)₂. A, Solid line: AnxA2^acryl; dotted line: (AnxA2^acryl-p11)₂. B, Solid line: AnxA2^acryl; dotted line: AnxA2^acryl LUV (PC/PS 75/25) L/P = 100 at pCa 2.7. Numerical values are shown in Table 2.

Fig. 5. Experimental fluorescence anisotropy decay of AnxA2^acryl and (AnxA2^acryl-p11)₂. A, AnxA2^acryl; B, AnxA2^acryl bound to LUV (PC/PS 75/25) (L/P = 100, pCa = 2.7); C, (AnxA2^acryl-p11)₂; D, (AnxA2^acryl-p11)₂ bound to LUV (PC/PS 75/25) (L/P = 100, pCa = 2.7). Experimental conditions as in Table 2.

Fig. 6. Correlation between membrane aggregation and fluorescence maxima. Maximum of the fluorescence emission spectrum of AnxA2^acryl (●) and turbidity (○) as a function of calcium concentration.

Fig. 7. Fluorescence emission spectrum of AnxA2^pyr. A: monomeric AnxA2^pyr; B) heterotetramer (AnxA2^pyr-p11)₂. (◊): 0.55 μM protein in buffer at 20 °C; (♦): 0.55 μM protein bound to LUV (PC/PS 75/25) L/P = 100 pCa3 at 20 °C; (●): 0.55 μM protein bound to LUV (PC/PS 75/25) L/P = 100 pCa3 at 2 °C. Excitation wavelength: 350 nm. Spectra normalized at 378 nm emission.

Fig. 8. Schematic topological model for AnxA2 organization in membrane bridges. In solution, the heterotetramer is stabilized by the interaction of S100A10 (in yellow) with the α-helix of the AnxA2 N-terminus (A). In membrane bridges the complex undergoes a subtle conformational change (B) which results in the increase of the AnxA2/p11interface dynamics. The data obtained with fluorescent probes on the Cys8 of the N-terminal tail (red beacon) revealed that the N-terminal domain in an “unfolded” state (C) change to a new conformational state when the protein bridges membranes. D and E show the single protein layer hypothesis and the two layer hypothesis respectively, in which the N-terminal domain lies very close to or in contact with the membrane. Our data disagree with these hypotheses (D) and (E), and support the model (F) in which the protein binds to the membrane via its convex face and protein contacts between the layers are formed at the concave face, allowing N-terminal tail interaction. The two N-terminal domains interact in membrane bridges, and are not in direct contact with the membrane.

AnxA2^acryl and (AnxA2^acryl-p11)₂ fluorescence quenching by iodide in solution and bound to membrane vesicles (LUV PC/PS 75/25)

Excitation wavelength: 392 nm, emission wavelength: 505 nm for AnxA2^acryl, 480 nm for AnxA2^acryl-LUV and (AnxA2^acryl-p11)₂. Protein concentration: 1 μM; L/P = 100. Estimated error of ± 10%.

The amplitude average lifetime <τ> was calculated as <τ> = Σα_iτ_i. and the bimolecular quenching constant as k_q = K_SV/<τ>.

MEM recovered excited state lifetime distribution of AnxA2^acryl and (AnxA2^acryl-p11)₂ in buffer and bound to phospholipid vesicles (LUV PC/PS 75/25)

Excitation wavelength: 392 nm, emission wavelength: 505 nm for AnxA2^acryl, 480 nm for AnxA2^acryl-LUV and (AnxA2^acryl-p11)₂. L/P = 100, L/P = 50; protein concentration: 0.5 μM. The standard deviation values were calculated from 2 to 5 measurements.

Amplitude average lifetime <τ> is defined as in Table 1.

MEM recovered rotational correlation time distribution AnxA2^acryl and (AnxA2^acryl-p11)₂ in buffer and bound to phospholipid vesicles (LUV PC/PS 75/25)

Experimental conditions as in Table 2.

The θ_i and β_i coefficients are respectively the values of the center and partial anisotropy of each rotational correlation time peak. The semi-angle ω_max of the wobbling-in-cone subnanosecond motion was calculated from (β₂ + β₃) / A₀ = [1/2cosω _max (1 + cosω _max)]², with ω_max = Arccos1/2[(1 + 8(Σβ(ns)_i/A₀)^1/2)^1/2−1], [49] with the intrinsic anisotropy A₀ = 0.370 measured in vitrified medium [48].