PMC

Effects of Munc18-1 on the conformation of the trans-SNAREpin detected by smFRET. A, fluorescent dye-labeled positions on syntaxin-1a and VAMP2. I203C or V241C of syntaxin-1a was labeled with Cy5 (light blue saw tooth circle), and Q33C or A72C of VAMP2 was labeled with Cy3 (red saw tooth circle). The Habc domain (white) of syntaxin-1a is depicted as broken to indicate the longer length than the SNARE motif (red). B, the TIRF microscope setup for the smFRET detection of the trans-SNAREpin conformation. The t-discs carrying Cy5-labeled syntaxin-1a (red and white) and SNAP-25 (green) were immobilized on the quartz surface through the biotin (yellow square) and streptavidin (purple cross) conjugation and then allowed to interact and form the trans-SNAREpin with the v-discs carrying Cy3-labeled VAMP2 (blue) with or without Syt1 (not shown). Distributions of the FRET efficiency for the NN and CC in the absence (top) or presence (bottom) of Munc18-1 without Syt1 (C) and with Syt1 (D), respectively. E, plot of the normalized FRET populations in the absence of Syt1 using the cut-off FRET efficiencies (E) of E ≤ 0.4 for low FRET, 0.4 < E < 0.72 for medium FRET, and E ≥ 0.72 for high FRET. F, the relative changes in the ratio of high to low FRET populations for the CC in the presence of increasing amount of Munc18-1 or 2 μm BSA without Syt1. G, docking ability of the CC in the absence and presence of 10 μm soluble VAMP2 (Vps, amino acids 1–94). H, plot of the normalized FRET populations in the presence of Syt1 using the cut-off FRET efficiencies (E) of E ≤ 0.4 for low FRET, 0.4 < E < 0.72 for medium FRET, and E ≥ 0.72 for high FRET. Error bars, S.D. of three independent experiments; movies were recorded for the analysis from more than five randomly selected screens in each experiment. *, p < 0.05, assessed using the two-sample Student's t test unless otherwise specified.

Qualitative consistency of SAXS and FRET. (a) Guinier plots for R17 at different concentrations of GdmCl and urea. Note that the primary data and fits are offset on the y-axis for clariy; broken lines show the fitted curves without offset to compare slopes. (b) Guinier fits carried out for R17 in 4.96 M GdmCl with different fitting ranges [0, q_max], where q_maxR_g is the number shown in the legend in (c). (c) R_g inferred from Guinier fits in (b), for different q_maxR_g. (d) Primary FRET data for unfolded R17 in GdmCl and urea. (e) End-end distance R inferred from R17 FRET in GdmCl, assuming Gaussian chain or SAW polymer models as labelled. (f) R_g inferred from R using fixed conversion factor for Gaussian chain or SAW.

(a) The root mean squared end-to-end distance (R_ee) as a function of GdmCl concentration for protein L. The average R_ee (black circles) and R for the subpopulation of the DSE [(red) squares] from simulations are shown. The values of R_ee inferred by solving Eq. by the standard procedure using the Gaussian chain, WLC, and SAW polymer models are shown for comparison as the top, middle, and bottom solid lines, respectively. The inset shows the relative errors between the exact and the values inferred using the FRET efficiency for R_ee vs GdmCl concentration. The top, middle, and bottom lines correspond to the Gaussian chain, WLC, and SAW polymer models, respectively. (b) Simulation results of the denatured state end-to-end distance distribution (P(R)) at 2.4M GdmCl [solid (red) squares] and 6M GdmCl [open (red) squares] and T=327.8 K are compared with P(R)s using the Gaussian chain, WLC, and SAW polymer models are also shown at 2.4M GdmCl (dashed lines) and 6M GdmCl (solid lines). The top, middle, and bottom lines correspond to the SAW, WLC, and Gaussian chain polymer models, respectively.

Denaturant-induced expansion of R17d and ACTR as seen from multiple methods. Panels a-d show the average end-to-end distances R in GdmCl and urea for R17d and ACTR. The corresponding radii of gyration are shown in panels e-h. Data are from a Guinier analysis of SAXS data with q_maxR_g ≤ 1.11 (green), from the analysis of FRET data with the Gaussian chain model as an upper bound (cyan) and a self-avoiding walk (SAW) as a lower bound (blue), from the ensemble refinement (red), and from all-atom molecular dynamics simulations of ACTR (black).