An engineered dimeric protein pore that spans adjacent lipid bilayers. Supplementary information. Bayley 1*

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1 An engineered dimeric protein pore that spans adjacent lipid bilayers Supplementary information Shiksha Mantri 1, K. Tanuj Sapra 1, Stephen Cheley 1, 2, Thomas H. Sharp 3 and Hagan Bayley 1* 1 Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK 2 Present address: Alberta Diabetes Institute, Department of Pharmacology, University of Alberta, Edmonton, T6G 2E1, Canada 3 Department of Physics, University of Oxford, Oxford OX1 3PU, UK 1

2 Supplementary figures and table Supplementary Figure S1. Hemolytic activity of HL mutants. (a) The hemolytic activities of two-fold serially diluted samples of WT, K237C/D8H6 (KC) and M113R/K237C/D8H6 (MR) 1 on rabbit erythrocytes were measured over 2 h in duplicates. The proteins were prepared by in vitro transcription and translation in the presence of [ 35 S]methionine. SDS-polyacrylamide gel electrophoresis and autoradiography showed that they were at closely similar concentrations. Both mutants lysed the blood cells leading to a decrease in optical density (O.D.) at 595 nm. (b) WT 7, KC 7 and KC (7) 2 were also examined. The concentrations of the samples examined were: WT 1, ~2 mg ml -1 ; WT 7, ~1 mg ml -1 ; KC 7, ~70 g ml -1 ; KC (7) 2, ~50 g ml -1. Therefore, well 5 of the WT sample is approximately equivalent in dilution to well 1 of the KC samples. Because well 5 of WT 1 shows 2

3 rapid hemolysis of the rabbit erythrocytes, we conclude that the other samples are inactive and uncontaminated with 1. 3

4 Supplementary Figure S2. Energy minimized cap-to-cap (7) 2. MD simulations of an aligned cap-to-cap (7) 2 structure linked by seven disulfide bonds were performed for 1 ns in 500 mm NaCl. (a) Cartoon representation of the energy minimized (7) 2 structure after a 1 ns all-atom MD simulation. The zoomed inset of the dimeric interface shows an absence of steric clashes (red disks). (b) The table shows values of overall short-range electrostatic energy, overall short-range Lennard-Jones energy and the number of hydrogen bonds at the interface of the two 7 units. Energy calculations were performed by using the GROMACS software version 4.0 and models were prepared in PyMOL. For the visualization of steric clashes, a Python script show_bumps (Thomas Holder) was used. 4

5 Supplementary Figure S3. Possible disulfide-linked structures of (7) 2. Schematic diagrams and space-filling models of all the possible (7) 2 structures. Each 7 unit is represented as a regular heptagon (grey) with orange vertices representing the cysteine locations. The structures can be placed in three classes: class 1, covalent-attached, but free to switch between misaligned and axially aligned forms by torsion about the disulfide bridge; class 2, a mixture of axially aligned forms and misaligned forms that cannot switch to an aligned form; class 3, all forms are axially aligned. (a) The (7) 2 structure with a single disulfide bond between the cis- 7 unit and the trans-7 unit. Rotation about the disulfide bond can lead to structures where the cis-7 unit partially occludes the trans-7 unit pore entrance and a fully aligned structure. Hence, structure 'a' is a class 1 structure. (b d) (7) 2 structures with two disulfide bonds between the cis-7 unit and the trans-7 unit. Structures 'b-d' are in class 2. There are also axially aligned forms with two disulfide bonds. The unitary ionic conductance values of structures a c would be similar to 5

6 7 because the cis opening of the trans-7 unit, which lies in the bilayer, is unobstructed by the cis-7 unit. In structure d, the cis-7 and the trans-7 units are connected through a water tunnel (yellow, visualized by MOLE 41 ) increasing the length of the pore. A water tunnel beginning in the unobstructed side of the trans-7 unit can also be seen (wheat-colored bulb). (e) The axially aligned cap-to-cap orientation is the only structure that is geometrically possible if the cis-7 and the trans-7 units are linked by three or more disulfides. Therefore, these structures are in class 3. Water-filled channels passing through the HL subunits were visualized in the cis-7 unit by using MOLE. Another possible path for ions is at the interface of the cis-7 and the trans-7 units. 6

7 Supplementary Figure S4. Particle size analysis of (7) 2. Individual (7) 2 molecules were observed by TEM after negative staining (Fig. 2d, e). Histogram showing distributions of single particles of (7) 2. Both, side and rings views (insets) were observed. The side views were elongated particles of average length 19.6±0.2 nm (mean ± s.e.m.) (n = 268) (peak 2). The ring-shaped particles had an average diameter of 7.8 ± 0.1 nm (mean ± s.e.m.) (n = 131) (peak 1). Insets show class averages of 309 single particles showing the side view and 224 single particles showing the ring view. Scale bar of insets: 20 nm. The dimensions of the single particles observed by TEM corresponded well with the modelled cap-to-cap (7) 2 structure (Fig. 1b). 7

8 Supplementary Figure S5. (7) 2 is resistant to proteolysis. SDS-PAGE gel showing that SDS-gel extracted (7) 2 (lane 1) and 7 (lane 5) were unaffected by proteinase K treatment (lanes 2 and 6 respectively). Proteinase K-treated (7) 2 dissociated into full-length 1 and (1) 2 upon heating at 95 o C for 10 min (lane 3). Proteinase K-treated (7) 2 gave 7 upon reduction with 3 M βme for 15 min, without heating (lane 4). Proteinase K-treated 7 yielded full-length 1 and another protein band (M app ~ 58,000) upon heating at 95 o C for 10 min (lane 6). On treating K237C 7 with proteinase K and subsequently with 3 M ME and heating, only full-length 1 was observed (data not shown). Again, the protein band (M app ~ 58,000) was not seen when WT 7 was treated with proteinase K, followed by heating (data not shown). Hence, the protein band (M app ~ 58,000) was attributed to a covalent complex of 1 (M app ~ 33,200) and proteinase K (M app ~ 29,000) linked through disulfide bonds. 8

9 Supplementary Figure S6. Open pore current (I o ) histograms of (7) 2 and 7. Histogram of I o of (7) 2 at (a) +50 mv (b) +100 mv and (c) +160 mv showed at least two populations of (7) 2 pores. At all potentials, the majority of the (7) 2 pores had a mean I o (red, fit 1) of ~65% of the I o of 7 (blue bars, fit 1'). These (7) 2 pores are believed to have axially aligned cap-to-cap structures (Fig. 1b, Supplementary Fig. S3e). The (7) 2 peak with the higher mean I o (green, fit 2) was close to the I o distribution for 7 (blue bars, fit 1') at the all the potentials tested, and is believed to arise from offset structures (Supplementary Fig. S3a-c). The values given are the mean I o ± s.d.. 9

10 Supplementary Figure S7. Phosphate (P i ) blocking of the MR (7) 2 mutant. In MR (7) 2 mutant pores with the axially aligned cap-to-cap arrangement of the cis-7 and trans-α7 units, (7) 2 should not be blocked by P i as the cap entrance of the trans-7 unit is occluded by the cis-7 unit, and P i binds only by entering through the cap domain 31. The I o histogram includes data for MR (7) 2 pores that were blocked by cis P i (filled blue bars) and for those that were not blocked (red outline bars). 17 of 65 pores were blocked by P i. 12 of the 17 pores had an I o value similar to that of 7. These pores are believed to have structures in which the entrance to the cap of the trans-α7 unit is not blocked by the cis-α7 unit (Supplementary Fig. S3a-d). 10

11 Supplementary Figure S8. Effect of freeze-thawing on P i binding and I o of MR (7) 2. The effect of freeze-thawing was studied by using 3-times (3x) freeze-thawed MR (7) 2. (a) I o histograms of 3x freeze-thawed pores that showed cis P i block (open bars) and pores that had been freeze-thawed once that showed cis P i block (blue bars), at +20 mv. P i binding was observed in 14 of 25 pores from the 3x freezethawed material, as compared to 17 of 65 pores of the MR (7) 2 freeze-thawed only once. (b) I o histograms of 3x freeze-thawed MR (7) 2 pores (open bars) and pores that had been freeze-thawed once (grey bars). The distribution of I o values for the 3x freeze-thawed pores was wider than that of the unfrozen pores. The mean I o value at +20 mv increased by ~2 pa after 3 freeze-thaw cycles. 11

12 Supplementary Figure S9. Comparison of I-V characteristics of individual 7 and (7) 2 pores, and (7) 2 inserted into a liposome. (a) I-V plot of (7) 2 with one barrel (that of the trans 7 unit) inserted in a planar lipid bilayer and the second barrel (the cis 7 unit) inserted into a liposome (open black triangles). The current through (7) 2 inserted into a liposome was lower than the current of 7 (open blue circles) and (7) 2 (open red squares) at both positive and negative potentials. (b) Plots of rectification ratios (I +V / I -V ) of 7, (7) 2, and (7) 2 in a liposome as a function of voltage. 12

13 Supplementary Figure S10. Molecular model of (7) 2 inserted in a planar bilayer and a porous liposome. The residual conductance of an (7) 2 pore inserted into both a planar bilayer and a liposome could be due to a leak current or additional (7) 2 pores in the liposome, which make it porous as depicted in the cartoon (not to scale). As the number of (7) 2 protein molecules present is much smaller than the number of liposomes in the cis compartment, it is unlikely that (7) 2 in solution will make the liposome completely porous. The consistent magnitude of the current blockades observed on liposome insertion and our theoretical calculations (Supplementary note 3) support the leak current hypothesis. 13

14 Supplementary Figure S11. Electrical model of (7) 2. (a, b) Representations of 7 and (7) 2 with electrical resistors. The cis- and trans-7 units of (7) 2 were considered as resistors in series, R c and R t, respectively. An additional leak resistance (R L ) was included in parallel with R c to account for additional conductive pathways in the structure. (c) Representation of 7 blocked with CD as having a resistor in series (R CD, orange). The switch represents the transient nature of the CD blockade. Electrical models of (7) 2 when CD is present on (d) the cis side, (e) the trans side, and (f) on both the cis and trans sides. 14

15 Supplementary Figure S12. Electrical circuits of (7) 2 inserted into both a planar bilayer and a liposome. (a) The residual current is attributed to a leak current (Supplementary Fig. S3e). In this case, the current passes through the leak resistor (R L ) and the trans-7 resistor (R t ), while the path through R c is blocked by the liposome (black switch open). (b) Circuit representing the reversible binding of CD to the trans-7 unit when (7) 2 is inserted into both a planar bilayer and a liposome. The curved arrow denotes reversible binding of CD. 15

16 Supplementary Table S1. Resistance and blockade values derived from the electrical model of (7) 2 compared to directly determined experimental values. The experimental current values of (7) 2 are supported by a model that considers the cis-7 and trans-7 units as resistors in series, represented by R c and R t (Supplementary note 3). The leak in the (7) 2 structure (R L ) was computed by using the experimentally determined mean I o values at -50 mv, +50 mv and +100 mv. At each applied potential, the value of the cis and trans CD blockades of (7) 2 were calculated by using R L, and the experimentally determined values of trans CD blockades of 7 at the corresponding potential. Applied Physical parameter or Value from Directly determined potential phenomenon electrical experimental value (mv) model (Average ± s.e.m.) +50 R t, R c, R d R t = 1.2 GΩ; R c = 1.2 GΩ; R d = 1.8 GΩ R L 1.2 GΩ cis CD blockade 14% of I o 15 ± 1% trans CD blockade 56% 55 ± 2% blockade by CD simultaneously 59% 61 ± 1% cis and trans blockade caused by liposome 25% 23 ± 1% insertion 16

17 blockade by trans CD during 49% 56 ± 1% liposome +100 R t, R c, R d R t = 1.1 GΩ; R c = 1.5 GΩ; R d = 1.7 GΩ R L 1.0 GΩ cis CD blockade 11% 13 ± 0.3% trans CD blockade 58% 54 ± 5% -50 R t, R c, R d R t = 1.2 GΩ; R c = 1.2 GΩ; R d = 1.8 GΩ R L 1.2 GΩ cis CD blockade 14% 19 ± 1% trans CD blockade 49% 48 ± 3% 17

18 Supplementary information discussion Supplementary note 1 Possible (7) 2 structures. Different combinations of disulfide bonds between the 7 units lead to different (α7) 2 structures (Supplementary Fig. S3). The 7 units of (7) 2 were modeled as regular heptagons in which the vertices represented the cysteine residues of each 1 subunit. To form a valid (7) 2 structure, the two heptagons were constrained to have at least one common vertex, i.e., a disulfide bond. According to this model, four structures other than the axially aligned cap-to-cap (7) 2 (Fig. 1b) are possible with a minimum of one and a maximum of two disulfide bonds between the 7 units. As compared to the fixed structures b-d (Supplementary Fig. S3) different conformations of disulfide bonds in structure a (Supplementary Fig. S3) can lead to alternative (7) 2 orientations including the axially aligned cap-to-cap structure (Fig. 1b). The aligned cap-to-cap (7) 2 structure can form with any number of disulfide bonds, from one to seven (Supplementary Fig. S3e). However, when 7 units or heptagons are linked at three or more positions, only the aligned cap-to-cap structure is geometrically possible. (7) 2 structures linked through different number of disulfides are predicted to yield different ratios of (1) 2 to 1 upon dissociation by heating at 95 C. If an aligned capto-cap arrangement of (7) 2 were formed with three or more disulfide bonds, the molar ratio of (1) 2 to 1 after heating would lie between 3 to 8 (three disulfide bonds) and 1 to 0, i.e., (1) 2 as the only product (seven disulfide bonds). Structure a (Supplementary Fig. S3), formed with one disulfide linkage, would yield (1) 2 and 18

19 1 in a molar ratio of 1 to 12 upon heating. Structures b-d (Supplementary Fig. S3) with two disulfide linkages would yield (1) 2 and 1 in a ratio of 1 to 5. Experimentally, upon heating (7) 2 at 95 C, (1) 2 and 1 were obtained in a molar ratio of ~1 to 2, from which we deduce that the average number of disulfides in (7) 2 was ~ 3.3. Hence, we conclude that the majority of the (7) 2 molecules were in an axially aligned cap-to-cap arrangement (Fig. 1b). The different structures of (7) 2 would also have different unitary conductance (G) values. G depends on the length (l) of a pore, its cross-sectional area (A) and the solution ionic conductivity (equation (S1)): To estimate the conductance of these protein pores, MOLE 41, an algorithm to visualize water-filled tunnels through protein pores was used. In structures a and b (Supplementary Fig. S3), there are no obstructions of the 7 pores by each other. Hence, the open pore current (I o ) of these structures would be similar to 7, given that just one of the two 7 units would span the lipid bilayer. Although there is minor obstruction of the trans-7 pore in structure c (Supplementary Fig. S3), the trans- 7 pore entrance is wider than the constriction in the barrel and the conductance of c is again expected to be similar to that of 7. In structure d (Supplementary Fig. S3), the two 7 units form a continuous water channel. Another water channel can also be visualized with an outlet at the cap-cap interface of the two 7 units. Unlike the tunnels in structures a, b and c, the length of the water tunnel in structure d is longer than that of 7 (Supplementary Fig. S3), suggesting that the conductance 19

20 of 'd' is less than that of 7. The axially aligned cap-to-cap (α7) 2, structure e (Fig. 1b), has twice the pore length of 7. Water tunnels are also found between the 1 subunits of the cis-7 half of structure e, but they are narrow (~3.7 Å in diameter) and unlikely to conduct hydrated ions. Therefore, it is likely that the proposed leak current, characterized by resistance R L (Supplementary note 3), occurs between the cis-7 and trans-7 halves of the (α7) 2 structure. Supplementary note 2 - P i block of freeze-thawed (7) 2. The effect of freezethawing MR (7) 2 was dramatic. When P i blockade was examined with a 3x freezethawed MR (7) 2 sample, we found that more than 50% of the pores (14 of 25) gave P i binding events compared to 26% of the pores from a sample that had been freezethawed once. The I o histogram of pores that were blocked by P i was broader in the case of protein that had been 3x freeze-thawed by comparison with the I o histogram from pores blocked by P i that had been freeze-thawed once (Supplementary Fig. S8a). Further, the mean I o of a 3x freeze-thawed sample was shifted by +2 pa at +20 mv compared to a sample that had been freeze-thawed once (Supplementary Fig. S8b). Multiple rounds of freeze-thawing would subject the protein to mechanical stress and thereby cause conversion of the aligned to misaligned (7) 2 structures. Freeze-thawing could also increase the extent of oxidation of the unreacted cysteine thiols. Thus (7) 2 could be locked as misaligned structures, which would explain the broad range of conductance values found with the 3x freeze-thawed (7) 2 aliquots. Supplementary note 3 - Electrical model of (7) 2. To interpret the observed electrical properties of (7) 2, an electrical model was constructed by considering the 20

21 cis- and trans-7 units as resistors in series, represented by R c and R t, respectively. In recordings of (7) 2 at +50 mv, the trans-7 unit is in the orientation of 7 at +50 mv, according to our sign convention, but the cis-7 unit is in the orientation equivalent to 7 at 50 mv. Therefore, the values of R c and R t were set to experimentally determined values of the unitary resistance of 7 at -50 mv and +50 mv, respectively, in 1 M KCl, 25 mm Tris.HCl, 50 µm EDTA, ph 8.0, R c = 1.2 GΩ and R t = 1.2 GΩ. The resistance of (7) 2 (R d ) at +50 mv was obtained from equation (S2). This differed from the experimentally observed value of R d, which was 1.8 GΩ. Hence, another resistor (R L ) was placed in parallel to R c (Supplementary Fig. S11b) to account for a leak in the structure between the two cap domains. By using equation (S3), the value of R L was determined to be 1.2 GΩ. Electrical model for cis and trans CD blocking. An additional resistance in series R CD (R CD, cis or R CD, trans ) was included to account for CD binding to the (7) 2 pore (Supplementary Fig. S11d-f). The values of R CD were determined from experimental data on the binding of CD to 7 (Supplementary Fig. S11c). At +50 mv, CD binding decreases the I o of α7 by 66% and at -50 mv by 58% (Table 1), i.e., the 21

22 current through the CD blocked pore (I res, CD trans ) was 34% of the I o for 7 at +50 mv and 42% (I res, CD cis ) of the I o for α7 at -50 mv. Based on these values, the resistance of the CD-blocked α7 pore was calculated at +50 mv (R CD +V ) and -50 mv (R CD -V ) to two significant figures by using equations (S4) and (S5) Hence, R CD, trans = 2.3 GΩ and R CD, cis = 1.7 GΩ. The expected magnitudes of blockades for cis and trans CD binding to (7) 2 can be calculated by using values of R L (equation (S3)), R CD (equations (S4) and (S5)), and equations (S6)-(S13) (Supplementary Fig. S11c-f). For cis CD binding, the resistance of the CD-blocked (7) 2 pore (R d, CD cis ) was calculated from equation (S6) and B CD cis, the blockade of I o, was thereby obtained from equation (S7). 22

23 R d, CD cis = 2.1 GΩ B CD cis = 14% For trans CD binding, B CD trans, the blockade of I o, was calculated from equations (S8) and (S9). R d, CD trans = 4.1 GΩ B CD trans = 56% When CD was present both and cis and trans sides, CD binding, blockade in I o (B CD cis-trans ) was computed using equations (S10) and (S11). 23

24 R d, CD cis-trans = 4.4 GΩ B CD cis-trans = 59% Current block due to liposome insertion. The residual conductance of (7) 2 after insertion of the cis-7 unit into a liposome might be explained by one of two scenarios (Supplementary Fig. S10). First, the cis-7 barrel could insert into a liposome already hosting (7) 2 pores. In this case, the cis-7 unit would still conduct. Assuming p additional (7) 2 pores insert into the liposome, the effective resistance (R d,lipo ) and current blockade (B lipo ) of the (7) 2 pore connecting the planar lipid bilayer and the liposome are given by equations (S12) and (S13) respectively. 24

25 This implies that the current blockade expected upon liposome insertion (B lipo ; equation (S13)) is a function of the number of pores inserted in the liposome (p). Based on the protein concentration and lipid concentration, we concluded that the number of free (7) 2 pores in solution that could insert in the liposome are much smaller than the number of liposomes. Hence, to explain the consistent current blockade of (7) 2 due to liposome insertion over different experiments, we evaluated the second scenario that could explain the residual conductance after insertion in a liposome. The second scenario assumes that the residual conductance of an (7) 2 pore in the planar bilayer after inserting into a liposome is due to a leak current within the (7) 2 pore (Supplementary Fig. S3e) and that the liposome is not penetrated by additional pores in the cis compartment. The apparent resistance, R d,lipo, of (7) 2 after insertion of the cis-7 unit into a fully insulating liposome is the sum of the leak resistance (R L ) and the resistance of the trans-7 unit (R t ), as given by equation (S14) (Supplementary Fig. S12a). Hence, the blockade in current can be estimated from equation (S13). B lipo = 25% When (7) 2 was inserted into both a liposome and a planar lipid bilayer, the effective resistance with CD bound from the trans side, R CD trans, lipo, and the value of the 25

26 trans CD block, B CD trans, lipo, were calculated by using equations (S15) and (S16) (Supplementary Fig. S12b). R CD trans, lipo = 4.7 GΩ B CD trans, lipo = 49% Electrical model at other potentials. The electrical model equations (S3) (S9) were used to calculate the leak conductance and the values of currents blockades by cis and trans CD at -50 mv and +100 mv (Supplementary information Table S1). At +100 mv, the experimental values of R t, R c and R d were 1.1 GΩ, 1.5 GΩ and 1.7 GΩ, respectively. R L was thus calculated to be 1.0 GΩ (equation (S3)). From equations (S4) and (S5), the values of R CD, trans and R CD, cis were 2.3 GΩ and 1.6 GΩ, respectively. The values of the cis and trans CD current blockades were computed to be 11% and 58%, respectively using equations (S6) (S9). The electrical values at -50 mv were computed similarly. 26

27 Supplementary methods Molecular modelling and graphics. All structures were rendered in PyMOL (Schrodinger) or VMD 42. The MOLE plug-in 41 for PyMOL was used to visualise water tunnels through the various (7) 2 pore structures (Supplementary Fig. S3, Supplementary note 1). This algorithm considers the centres of the van der Waal's spheres of the all protein atoms to create a Voronoi mesh. The edges of the Voronoi cells farthest from the central atoms are ranked based on an empirical cost function and connected to compute optimal egress paths or channels through the protein. For Supplementary Fig. S3b-e, the starting point for tunnel exploration was the centre of the trans-α7 unit. The number of tunnels to be searched for in each structure was fixed at a minimum value of 3. Protein preparation. The mutation K237C was introduced into the wild-type HL gene in a pt7 vector with an ampicillin resistance gene 38 by PCR-based site-directed mutagenesis followed by homologous recombination of the PCR products in XL10- Gold ultracompetent E. coli cells (Agilent). The vector also encoded a D8H6 tag at the C-terminus of the HL gene to aid protein purification by Ni 2+ -NTA chromatography. The M113R mutation was introduced into the pt7 K237C/D8H6 plasmid by a further round of PCR-based site-directed mutagenesis. Rosetta (DE3) plyss competent cells (150 L, Novagen) were transformed with ~2 g of the HL K237C/D8H6 or HL M113R/K237C/D8H6 plasmid DNA by first incubating the cells on ice for 30 min, followed by heat shock at 42 C for 30 s. The 27

28 transformed cells were plated on ampicillin (200 g ml -1 ) and chloramphenicol (25 g ml -1 ) selective LB-agar plates. A single colony was picked and inoculated into 100 ml of Terrific broth containing ampicillin (200 g ml -1 ) and chloramphenicol (25 g ml -1 ), which was then grown at 37 C for ~16 to 18 h. 5 ml of this culture was then used to inoculate 100 ml of Terrific broth containing the antibiotics, which was grown at 37 C. At OD ~0.6, the culture was induced with isopropyl β-d-1- thiogalactopyranoside (IPTG) (0.5 mm) and grown for a further 22 h at 20 C. HL K237C/D8H6 was purified under non-denaturing conditions. The culture was centrifuged at 3400 xg for 25 min and the cell pellet was suspended in lysis buffer (50 mm Tris.HCl, 200 mm NaCl, 1% v/v Triton X-100, 10 mm imidazole, 1.2 µl benzonase (Merck), 1 mm MgCl 2, 0.1 mm tris(2-carboxyethyl)phosphine (TCEP), ph 8.0). The lysate was sonicated for 5 min in 30 s pulses with 30 s pauses, and then centrifuged at 3,400 xg for 15 min. The supernatant was loaded onto a preequilibrated Ni 2+ -NTA agarose column (2 ml, Qiagen). The column was washed three times with 15 ml of wash buffer (50 mm Tris.HCl, 500 mm NaCl, 30 mm imidazole, 0.2% v/v Triton X-100, ph 8.0). The proteins were eluted with 12 ml of elution buffer (50 mm Tris.HCl, 500 mm NaCl, 0.2% v/v Triton X-100, 500 mm imidazole, ph 8.0). All steps were performed on ice or at 4 C. A mixture of the HL proteins 1, (1) 2, 7 and (7) 2 eluted together from the Ni 2+ - NTA columns. To separate 1, (1) 2, α7 and (7) 2, SDS-PAGE of the eluate in Laemmli sample buffer 43 was performed. The eluates were loaded as duplicates on a 8% (w/v) polyacrylamide gel (length 16 cm, thickness 1 mm), which was run at +60 V in TGS (25 mm Tris.HCl, 192 mm glycine, 0.1% w/v SDS) running buffer for 16 to 28

29 20 h at 25 C. After electrophoresis, the gel was split into an analytical half which was stained using Coomassie Blue to detect the bands of α7 and (7) 2 and a preparative half which was sealed with cling-film and kept at 4 C. Gel pieces containing α7 and (7) 2 were excised from the preparative gel using the analytical gel as a reference, and crushed in 500 L of 10 mm Tris.HCl, 1 mm EDTA, ph 8.0 (TE). After incubation for 16 h at 4 C, the crushed gel suspension was loaded onto spin-filtration columns (Rainin) and the protein fractions were eluted by centrifugation at 25,000 xg for 15 min at 4 C. The concentration of (7) 2 was determined by Bradford assay in triplicate 44. The molar ratio of 1 and (1) 2 after heating (7) 2 at 95 C for 10 min was estimated from the band intensities after Coomassie blue staining of SDS polyacrylamide gels by using Quantity One 1-D Analysis Software (Bio-Rad). HL M113R/K237C/D8H6 was purified under denaturing conditions. The transformation protocol and the growth conditions of the Rosetta (DE3) plyss cells expressing HL M113R/K237C/D8H6 were the same as used for HL K237C/D8H6. The pellet from the cell lysate was resuspended in 8 M urea, and centrifuged at 13,000 xg for 30 min at 4 C to remove insoluble debris. Buffer containing 8 M urea was used in the subsequent purification step with Ni 2+ -NTA agarose (Qiagen, a derivative of Sepharose CL-6B, which is stable in urea). The first three eluate fractions (5 ml each) were combined and dialysed overnight against 50 mm Tris.HCl, 500 mm NaCl, 0.2% v/v Triton X-100, ph 8.0, by using a dialysis membrane (cut-off 3500 Da, Spectrum) to remove the urea and refold the protein. The dialysed protein was concentrated ten-fold with 10 kda cut-off centrifugal filter columns (Millipore). M113R/K237C/D8H6 α7 and (7) 2 were further purified from 8% 29

30 SDS polyacrylamide gels in the same way as the K237C/D8H6 proteins described above. Statistical analysis. Unless mentioned, n denotes the number of events used to compute the mean. N denotes the number of independent experiments, i.e., experiments done over different days. The I o histograms of α7 and (7) 2 were fit with Gaussian functions. The coefficients of the Gaussian functions are reported as mean ± s.d. (equation (S17)). For all other data, the mean ± s.e.m. values have been reported. In experiments where only a fraction of the (7) 2 pores displayed a certain behaviour (for example, P i binding) while the other (7) 2 pores did not, the (7) 2 pores could be divided into two classes (equivalent to a binomial distribution). The proportion of (7) 2 pores in each class has been reported with a 95% confidence interval. The confidence interval of a binomial distribution is given by equation (S18). where, p is the proportion of successes in n attempts. For a 95% confidence interval, z 1- /2 is equal to

31 Supplementary references 41. Petrek, M., Kosinova, P., Koca, J. & Otyepka, M. MOLE: A Voronoi diagrambased explorer of molecular channels, pores, and tunnels. Structure 15, (2007). 42. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J Mol Graph 14, 33-8, 27-8 (1996). 43. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, (1970). 44. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, (1976). 31