Results: 4

1.
Figure 3

Figure 3. From: Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex.

Syt1 binds to SNARE complexes reconstituted in 100% phosphatidylcholine (PC) bilayers more strongly at decreased ionic strength of the buffer. (a) The number of Syt1 bound per 5000 μm2 of PC bilayer containing equal reconstitutions of SNARE complexes is increased at lower ionic strength (circles). Ca2+ further enhances the binding (triangles). Little binding occurred in the absence of SNARE complex (squares). Those few molecules that bound to protein free bilayers diffused freely in the plane of the bilayer. Syt1 bound to SNARE complex containing bilayers exhibit much lower diffusivity. (b) Identical experiments using Syt1 binding to bilayers containing syntaxin– SNAP-25 binary complexes also show increased binding at decreased ionic strength, but the presence of Ca2+ has a smaller effect than for the ternary SNARE complex. The densities of the bilayer-incorporated complexes are 0.4 ternary SNARE complex/μm2 (a) and 0.7 binary SNARE complex/μm2 (b) (Online Methods). Considering the concentration of Syt1 was below 20 nM for all incubations, Syt1 binding to the protein containing surfaces is not saturating.

Ucheor B. Choi, et al. Nat Struct Mol Biol. ;17(3):318-324.
2.
Figure 2

Figure 2. From: Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex.

smFRET shows that Syt1 conformations are dynamic. (a,b) Experiments as in Fig. 1c were repeated three to five times for each indicated condition. The average values of the center and width of the Gaussian fit to the FRET efficiency distribution peak are shown. Error bars indicate two standard deviations. (c) Application of the bi-functional NHS-ester crosslinker (BS3) before liposome encapsulation to Syt1 C2AB with labels attached to residues 154 (C2A) and 383 (C2B) narrows the width of the smFRET efficiency distribution. (d) Control experiments using dye labels that both reside in C2A (residues 140 and 154) for encapsulated or SNARE bilayer immobilized experiments at 50 mM NaCl, and Ca2+ or EDTA, as indicated. (e) smFRET donor and acceptor intensity trajectories in control experiments as in (d) without (top) and with (bottom) Ca2+, using liposome encapsulation (left) and supported bilayers (right). No transitions other than photobleaching were observed. (f) Percent of the molecules with at least one transition between non-zero FRET efficiency levels during the 90 second observation period for Syt1 with labels attached to residues 154 (C2A) and 383 (C2B) under indicated conditions.

Ucheor B. Choi, et al. Nat Struct Mol Biol. ;17(3):318-324.
3.
Figure 1

Figure 1. From: Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex.

smFRET reveals Syt1 conformations. (a) Surface immobilization schemes using liposome encapsulation (left) or supported lipid bilayers (right). Sx is syntaxin; S25 is SNAP-25; Sb is synaptobrevin; Syt1 is synaptotagmin. 50 mM NaCl and 1 mM Ca2+ or 1 mM EDTA were used for all experiments. (b) Intensity time traces for single molecules of Syt1 with labels attached to residues 154 and 383. Note the transition between mid and high FRET in the upper left graph. The sudden downward transitions to zero acceptor intensity are due to dye photobleaching. (c) Histograms of smFRET measurements using Syt1 with donor in one C2 domain and acceptor in the other (label pairs are indicated on the left edge). Arrows indicate the peak value (designated E in the figure) from Gaussian fits. For comparison, converting Cα separations in the Syt1 crystal structure 22 to FRET using Ro=5.55 yields: 0.99 (residues 154 and 383), 0.77 (residues 154 and 396), 0.95 (residues 189 and 396), 0.75 (residues 254 and 396). See also Supplementary Fig. 9.

Ucheor B. Choi, et al. Nat Struct Mol Biol. ;17(3):318-324.
4.
Figure 4

Figure 4. From: Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex.

smFRET-derived model of the Syt1–SNARE complex. (a) smFRET efficiency time traces and (b) smFRET histograms of labeled Syt1–SNARE complex. We used combinations of six different single donor label sites in Syt1 (indicated in the left column) (residues 140 and 154 in C2A; residue 269 in the linker; residues 368, 383, and 350 in C2B) and single acceptor label sites in the SNARE complex (indicated at the top) at the N-terminus (SNAP-25 residues 20 and 139), C-terminus (SNAP-25 residues 76 and 197), and the center (synaptobrevin residues 61) of the SNARE complex. Circles indicate the smFRET efficiency values from which distances were derived for the docking calculations. (c) Shown is the best model (in terms of the rms distance range deviation (rmsdrd) from the borders of the restraining square-well potential, rmsdrd=0.38 nm, see Supplementary Note). Coordinates of the model are available as Supplementary Data. Three rotated views are displayed. C2A is colored orange, C2B yellow, SNAP-25 green, synaptobrevin blue, and syntaxin red. The conserved arginine residues 398 and 399 in C2B are shown as red sticks, and the Ca2+-binding loops are colored red. The docking calculation of the syt1–SNARE complex involves 34 independent distance measurements constraining 12 independent parameters in possible models (3 rigid bodies × (3 rotation + 3 translational degrees of freedom) − (6 over-counting for coupled motions in global translations/rotations) = 12 degrees of freedom).

Ucheor B. Choi, et al. Nat Struct Mol Biol. ;17(3):318-324.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk