Figure 5. Oligomerization states for arc- and ring-shaped assemblies of suilysin.

(A) The arc-length distribution of wild-type suilysin displays a broad peak for arcs that contain between 15 and 30 monomers, and a smaller, sharp peak for complete rings (37-mers). (B) Arc-length distribution for the disulphide-locked suilysin prepore intermediate. (C) For the disulphide-locked mutant incubated in the presence of DTT (pore-state), the arc-length distribution is practically identical to the distribution for the prepore-locked intermediate. (D) Calculated arc-length distributions for a simple model of kinetically trapped oligomerization, with C = 2000 monomers per square micron (see ‘Materials and methods’). The peak of the arc-length distribution shifts from smaller to larger oligomers on increasing the ratio between the rate constants for monomer association (k_a) and monomer binding to the membrane (k_b). Vertical scale bar: 40 counts. Grey, dashed lines in A–C denote fits of the experimental data with the oligomerization model, yielding k_a/k_b = 0.893 ± 0.008 µm² (A); 0.438 ± 0.012 µm² (B); 0.425 ± 0.012 µm² (C). Numbers in brackets in A–C indicate the estimated total number of monomers per square micron. The experimental data here are based on negative-stain EM images on monolayers of egg PC:cholesterol (45:55%), incubated at 37°C.

DOI: http://dx.doi.org/10.7554/eLife.04247.017

Figure 5—figure supplement 1. Sequential addition of wild-type suilysin in the pore state.

(A–D) Sequence of AFM images in the same area, showing the effect of sequential injections of wild-type suilysin (WT-SLY) in the solution above the supported lipid bilayer (egg PC:cholesterol, 67:33%) at 27°C. The overall increase in the number of arcs and rings in the pore state can be readily observed while individual arcs in the pore state can be tracked and characterized following each injection. (E) After the third injection, the open-ended arc still persists and the length of the arc does not increase further even as more arcs and rings have assembled in the vicinity of the open-ended arc. (F) After the fourth addition of WT-SLY, the open-ended arcs of SLY are still prevalent and the newly formed arc is interlocked with the arc already present from the previous addition of toxin. (G) The results show that while the total number of monomers in the pore state increases after each WT-SLY addition, the arc length distribution remains unchanged. This implies that after each monomer addition, new arcs (and ring complexes) are formed that do not oligomerize with arcs already in the pore state. Thus suilysin oligomers can only assemble in the prepore state.

DOI: http://dx.doi.org/10.7554/eLife.04247.018