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1 Ultrafast proton transport in sub-1-nm diameter carbon nanotube porins Ramya H. Tunuguntla, 1 Frances I. Allen, 2 Kyunghoon Kim, 1, Allison Belliveau, 1 Aleksandr Noy 1,3, * * Correspondence to AN: noy1@llnl.gov Table of contents: S1. Calculating Liposomal Buffering Capacity S2. Determining Proton Flux and Proton Conductance of CNT porins S3. Experimental Results and Fitting Analysis of CNT porin proton conductance in presence of Ca 2+ ions S4. Supplementary References S5. Supplementary Figures NATURE NANOTECHNOLOGY 1

2 S1. Calculating Liposomal Buffering Capacity The internal buffering capacity of the liposomes is calculated by adding aliquots of a strong acid (1 M HCl) to LUVs in 10mM HEPES buffer solution at ph 7.5 to calibrate the change in ph to the change in fluorescence (Supplementary Fig. 1a). Subsequently, we used this calibration to quantify the lumenal acidification of the liposomes when they were exposed to step additions of a weak acid (10 mm sodium acetate) (Supplementary Fig. 1b). Since our initial ph was not equal to the pk a of the proton donor molecule, HEPES (pk a = 7.55), we used the Henderson-Hasselbalch equation to determine the initial concentrations of the conjugate base and acid in the HEPES buffer solution. pppp = ppkk % + llllll [,- ] [/,] (1) We used equation 1 to calculate the amount of acid required to deplete the conjugate base and result in the decreased internal ph. Finally, we used equation 2 to obtain our buffer capacity value (BCV) as M LL 23 pppp 23 : BBBBBB = 789:;< 9= %>?@ (89BCD) F9B:8C I/ (2) S2. Determining Proton Flux and Proton Conductance of CNT porins We calculated the proton flux across the vesicle walls from Eq. 1 in the main text, using the measured rate of ph change, the measured buffer capacities, and the sizes of the vesicles (182 ± 17 nm for LUVs, and 238 ± 38 nm and 191 ± 29 nm for CNT-LUVs, for 1.5 nm and 0.8 diameter porins, respectively) as inputs. The apparent proton flux, J LUV, for LUVs is calculated to be ± mol * s -1 * cm -2. When CNT porins are incorporated into the LUVs the proton flux, J CNT-LUV increases to 5.76 * ± 7.80 * mol * s -1 * cm -2 and ± mol * s -1 * cm -2 for LUVs containing porins of 1.5 nm and 0.8 nm diameters, respectively. The measured background proton permeability, P + H, through the control DOPC LUV membranes was cm*s -1 (where ΔC ~ mol * cm -3 ). This value compares well with previously reported values of cm s -1 by Deamer 1 and cm s -1 by Rossignol 2 and the range of cm s -1 reported by Biegel and Gould 3. 2 NATURE NANOTECHNOLOGY

3 SUPPLEMENTARY INFORMATION For the CNT-LUV measurements the proton flux that goes through the CNT pore openings is equal to the difference between the flux through CNT-LUVs and the flux through control LUV membranes. When we scale this difference to the area of the CNT mouth opening, we obtain the following relationship: JJ KLM AA KLM = JJ KLM2OPF AA KLM2OPF JJ OPF (AA KLM2OPF AA KLM ) (3) where J CNT, J CNT-LUV, and J LUV are proton fluxes through the CNT porin and LUVs containing CNT porins and empty LUVs, respectively; A CNT and A CNT-LUV are the areas of the CNT mouth opening and of the LUVs containing CNT porins, respectively. Using equation 3, we obtained J CNT, the flux through CNTs porins, to be and mol s -1 cm 2 for the wide and narrow porins, respectively. We can then use the measured number of CNT porins in a liposome to calculate the single channel conductance for a CNT porin (Eq. 4): CCCCCCCCuuuuuuuuuuuuuu pppppp CCCCCC = JJ KLM AA I9]C ^ ( I/ _._ab) (4) where F is the Faraday s constant, A pore is the area of a CNT pore, and ΔpH is the driving force, converted to volts using the relationship that 1 ph unit is equivalent to V. S3. Experimental Results and Fitting Analysis of CNT porin proton conductance in presence of Ca 2+ ions At high Ca 2+ ion concentrations the proton conductance of CNT porins reaches a saturation value. This effect can be seen more clearly when the data is plotted as CNT porin resistance as a function of Ca 2+ ion concentration (Fig. 4b). Control experiments where we added increasing concentrations of Ca 2+ to a sample of LUVs (Supplementary Fig. 2) with ph equilibrated to 6.9 both inside and outside the vesicle lumen, did not show any changes in the dye fluorescence, indicating that the presence of CaCl 2 does not affect the measurement by changing the pyranine dye fluorescence intensity. Thus, we assumed that the increased proton current resistance of the CNT porins is the result of the Ca 2+ ions adsorption at the pore mouth. In this case we can describe this process using a Langmuir isotherm (Eq. 5): RR = RR _ + RR 8%d ek 3feK (5) NATURE NANOTECHNOLOGY 3

4 where R 0 is the pore resistance at 0 mm CaCl 2, R max is the maximum resistance at the saturation levels of CaCl 2, b is Langmuir adsorption constant that describes the affinity to the binding sites, and C is CaCl 2 concentration. This model allows us to estimate b, which is essentially the saturating Ca 2+ concentration at the mouth of the pore, C sat. S4. Supplementary References 1 Nichols, J. W., Hill, M. W., Bangham, A. D. & Deamer, D. W. Measurement of Net Proton- Hydroxyl Permeability of Large Unilamellar Liposomes with the Fluorescent ph Probe, 9- Aminoacridine. Biochim. Biophys. Acta 596, (1980). 2 Rossignol, M., Thomas, P. & Grignon, C. Proton permeability of liposomes from natural phospholipid mixtures. Biochim. Biophys. Acta 684, (1982). 3 Biegel, C. M. & Gould, J. M. Kinetics of Hydrogen-Ion Diffusion across Phospholipid Vesicle Membranes. Biochemistry 20, , (1981). 4 NATURE NANOTECHNOLOGY

5 SUPPLEMENTARY INFORMATION S5. Supplementary Figures Supplementary Fig. 1 (a) A calibration curve showing the changes in the dye fluorescence at a given intravesicular ph. Blue open-circle data points represent averaged values from four separate measurements (red, green, black, and yellow) (b) The change in intravesicular ph after repeated stepwise additions of 10 mm sodium acetate. These data were used for calculating the buffer capacity of the vesicles. Dashed lines represent the best linear fit to the data. NATURE NANOTECHNOLOGY 5

6 Supplementary Fig. 2 Addition of CaCl 2 to the samples containing pyranine-entrapped CNT LUVs (equilibrated at ph 7.5) does not affect the dye emission properties or the calibration for converting fluorescence emission to ph change for CNT-LUVs. The change in fluorescence after adding the CaCl 2 solution is identical to the fluorescence change resulting from the dilution of the existing solution in the fluorescence cuvette. 6 NATURE NANOTECHNOLOGY

7 SUPPLEMENTARY INFORMATION a 10 b Counts CNT diameter (nm) c Intensity(au) nm plot profile Gaussian fits sum of Gaussian fits -10 background peak fit region background Distance (nm) Supplementary Fig. 3 TEM analysis of CNT diameters. (a) Histogram of the CNT diameters measured from TEM images of partially-cut CNTs solubilized with DOPC lipid (N=20). To obtain good dispersion of the CNTs in the imaging samples, we stopped the sonication procedure after 5 hours (instead of the 16 hour duration used to prepare CNTPs). Black solid line indicates Gaussian fit to the data. (b) Representative TEM image of partially-cut CNT sample used for diameter determination. Scale bar: 10 nm. Dashed black line indicates the image region that was used to generate the TEM line profile for fitting. (c) Illustration of the fitting procedure used to obtain the CNT diameter from TEM line profiles. See Methods for procedure description. NATURE NANOTECHNOLOGY 7

8 Supplementary Fig. 4 TEM analysis of the CNTPs. (a) Histogram showing lengths of the CNTPs before membrane insertion (blue bars) as measured from TEM images. Solid line indicates a fit to a log-normal distribution (mean value: 11.7±2.3 nm, N=26). (b) Representative TEM images (i-iv) of isolated CNTPs. Red circles indicate locations of CNTPs. 8 NATURE NANOTECHNOLOGY

9 SUPPLEMENTARY INFORMATION Supplementary Fig. 5 Cryogenic TEM images of CNTPs inserted into lipid bilayers. (a) Widefield image of liposomes with inserted CNTPs. Green frames (i-vi) indicate regions with visible CNTPs. (b-e) Zoom-in images of the areas (i-vi) showing CNTPs inserted in the lipid bilayer. Red frames indicate locations of the CNTPs. All scale bars: 10 nm. NATURE NANOTECHNOLOGY 9

10 Supplementary Fig. 6 (a, inset) Schematic illustrating CNT-LUV porins labeled with a 1.4 nm gold nanoparticle conjugated to a fluorescently labeled antibody fragment. (a) UV-VIS absorbance spectrum of LUVs containing Au-FITC nanoparticle conjugated CNTPs. Absorbance value at 494 nm (after background subtraction from LUV absorbance) allows determination of FITC dye concentration in a given sample batch. (b) UV-VIS absorbance spectrum of lipid-ferrothiocyanate compound used to evaluate the lipid amount in the sample. (c-d) Cryogenic transmission electron micrographs of CNT porins with a Au-FITC nanoparticle label attached to the protruding ends of the CNTPs. (c) (Left) Large area view of CNT-LUVs on TEM grid. (Middle) Magnified view of the green-boxed area. Dashed green boxes highlight CNTPs with Au-nanoparticle label and (Right) the positions of the nanotubes and gold labels are marked with red lines and yellow circles, respectively. (d) CNTP in lipid bilayer. Unprocessed image is on the left, and the image overlaid with markers to highlight the position of the CNT porin, gold label is on the right. All scale bars: 10 nm NATURE NANOTECHNOLOGY

11 SUPPLEMENTARY INFORMATION Supplementary Fig. 7 Proton transport through gramicidin A pores. (a) Normalized fluorescence intensity changes (I/I 0 ) as a function of time for proton transport through gramicidin A pores inserted into the LUVs. (a, inset) Magnified initial region of ph gradient dissipation kinetics through gramicidin A pores (blue) and in DOPC liposomes (black) in ph range from 7.5 to 6.9. (b) The natural logarithm of the measured proton transport rates plotted against the reciprocal of the temperature. The Arrhenius activation energy (E A ) was determined from the slope of the linear regression to the data. 11 NATURE NANOTECHNOLOGY 11

12 Supplementary Fig. 8 Proton permeability of CNTPs is independent of the direction of the proton gradient. Whether proton flux was initiated by the acidification of the external buffer (acid jump, blue trace) or by alkalinization (base jump, red trace), the transport kinetics produces the same proton permeability coefficients. 12 NATURE NANOTECHNOLOGY