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1 Internal Dynamics of a Supramolecular Nanofiber Julia H. Ortony 1, Christina Newcomb 1,2, John B. Matson 1, Liam C. Palmer 3, Peter E. Doan 3, Brian M. Hoffman 3, and Samuel I. Stupp 1,2,3,4 1 Institute for BioNanotechnology in Medicine, Northwestern University, 303 E. Superior St., Suite , Chicago, IL 60611, USA. 2 Department of Materials Science and Engineering, Northwestern University, 2220 Campus Drive, Evanston, IL 60208, USA. 3 Department of Chemistry, Northwestern University, 2220 Campus Drive, Evanston, IL 60208, USA. 4 Department of Medicine, Northwestern University, 251 East Huron Street, Chicago, IL 60611, USA. Present address: Department of Chemistry, Virginia Tech, Blacksburg, VA 24060, USA. * s-stupp@northwestern.edu 1. PA Synthesis, Purification, and Sample Preparation 1.1 PA Synthesis. Syntheses of PAs were performed either manually or on a CEM Liberty microwaveassisted peptide synthesizer from Rink Amide MBHA resin using standard solid phase synthesis conditions. For each manual coupling, 4 equiv of Fmoc-protected amino acid was added using 4 equiv HBTU and 6 equiv DIEA in 20 ml DMF (HBTU = O-benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate; DIEA = N,N-diisopropylethylamine). Fmoc removal was accomplished with 30% piperidine in DMF. For each microwave-assisted coupling, 5 equiv of Fmoc-protected amino acid in DMF was added with 5 equiv HBTU in DMF and 10 equiv DIEA in NMP (NMP = N-methyl-2- pyrrolidone). Fmoc removal was accomplished using a solution of 20% piperidine in DMF and 0.1 M HoBt (HoBT = hydroxybenzotriazole). Default settings for microwave power and duration were used. The palmitic acid tail was added using the same coupling conditions unless otherwise noted. PAs were cleaved from the resin using a peptide cleavage solution of 95% TFA, 2.5% TIPS and 2.5% H 2 O. Concentration of the cleavage solution in vacuo and precipitation of the residue into cold Et 2 O afforded the crude product, which was purified by preparative HPLC. For synthesis of PA 2, the methylated valine reside was coupled and deprotected as previously described. 1 5 equiv. of Fmoc-Val(Me)-OH (EMD), 5 equiv. PyAOP and 10 equiv DIEA in DMF (3mL/g resin) were reacted in a shaker vessel with resin at room temperature for 1 h (PyAOP = 7-azabenzotriazol-1-yloxy)- tris(pyrrolidino)phosphonium hexafluorophosphate). After 1 h, an additional 2.5 equiv PyAOP was added to the vessel and allowed to react for an additional hour. Deprotection of the methylated valine residue was performed with two washes of 5:5:20:70 piperidine:dbu:toluene:dmf 10 min each. (DBU = 1,8- diazabicyclo[5.4.0]undec-7-ene) PA 1. Synthesis was performed on the CEM Liberty synthesizer as described above. The product was purified by HPLC. MS (M+H): calc d: ; found: PA 2. Synthesis was performed by hand as described above. The product was purified by HPLC. MS (M+H): calc d: , found: NATURE MATERIALS 1

2 PAs 1a, 1b, and 1c. Syntheses were performed manually as described above, using Fmoc-TOAC for the spin-labeled residue. PA 1a: MS (M+H): calc d: ; found: PA 1b: MS (M+H): calc d: ; found: PA 1c: MS (M+H): calc d: ; found: PAs 1d and 1e. A peptide with the sequence V 2 A 2 E 2 with a free N-terminus was synthesized as described above. The product was lyophilized from water after precipitation from Et 2 O but not further purified. The alkyl tails as their carboxylic acids (5-DOXYL-stearic acid free radical and 16-DOXYL-stearic acid free radical for 1d and 1e, respectively) were first converted to their NHS esters by reaction with NHS (1.05 equiv) and DCC (1.05 equiv) in dioxane at room temperature. After overnight stirring, the reaction mixture was filtered and diluted with CH 2 Cl 2 (10 ml) then washed with saturated aqueous NaHCO 3 (10 ml), H 2 O (10 ml) and brine (10 ml). The organic layer was dried over Na 2 SO 4 and concentrated to yield an orange, waxy solid. This crude solid was dissolved in 1 ml DMSO and added to the V 2 A 2 E 2 peptide. After standing overnight, the reaction mixture was diluted with 0.1% NH 4 OH and purified by HPLC. PA 1d: MS (M+H): calc d: ; found: PA 1e: MS (M+H): calc d: ; found: PA Purification. Purification by preparative-scale HPLC was carried out on a Varian Prostar 210 HPLC system, eluting with 2% ACN to 100% ACN in water on a Phenomenex C18 Gemini NX column (150 x 30 mm) with 5 μm pore size and 110 Å particle size. 0.1% NH 4 OH was added to both mobile phases to aid PA solubility. Product-containing fractions were confirmed by ESI mass spectrometry (Agilent 6510 Q-TOF LC/MS), combined, and lyophilized after removing ACN by rotary evaporation. 1.3 Sample Preparation. Careful sample preparation procedures were carried out to ensure uniform coassembly. Mixtures of PA 1 with each of PAs 1a-1e were sonicated in 5% NH 4 OH for 2 h at rt to prevent reduction of the nitroxide radical. 2 After sonication, the solutions were equilibrated for 1 h, then lyophilized to a powder. Each powder sample was resuspended in pure water at 2 wt%, then diluted to 1 wt% for a final salt concentration of 100 mm CaCl 2. PA 1/2 was prepared by mixing equimolar solution of PAs 1 and 2 at a 1:1 ratio, then combining with each of PAs 1a-1e as described above. To preclude the possibility of exchange broadening in the EPR spectra that results from the interaction of adjacent spin labels, EPR spectra of PA 1 with each of PA 1a-1e were acquired as a function of percent spin label (PA 1a-1e) incorporation. Fig. S2 shows a representative set of EPR spectra of the spin label ratio series. When the PA nanofibers are composed of 0.4% PA 1c in 99.6% PA 1, no spectral broadening is observed. At spin label concentration of 1.0% and higher, spectral broadening is observed. Thus, the percent incorporation of PAs 1a-1e in PA 1, and PA 1/2 coassemblies, was held at 0.4% for all spectra presented in Fig NATURE MATERIALS

3 Figure S1. EPR spectra of PA 1/1c composite nanofibers with different concentrations of spin labeled PA (PA 1c) from 0.2% to 5.0%. As the ratio of PA 1c increases, spectral broadening is observed due to distance-dependent electron-electron exchange interactions. Sweep width: 150 G. 2. Structural characterization of supramolecular assemblies 2.1 Cryogenic Transmission Electron Microscopy (cryo-tem) Figure S2. Cryogenic TEM of PA solutions used for EPR experiments at 0.1% (w/v) diluted from a 1% (w/v) solutions. All samples form high aspect ratio nanofibers regardless of the position of the spin label. NATURE MATERIALS 3

4 A mixture of PA 1 with spin labeled (a) PA 1a, (b) PA 1b, (c) PA 1c, (d) PA 1d, (e) PA 1e, and (f) PA 1/2. Scalebars: 200 nm. 2.2 Small Angle X-ray Scattering (SAXS). Measurements were performed using beam line 5ID-D, in the DuPont-Northwestern-Dow Collaborative Access team (DND-CAT) Synchrotron Research Center at the Advanced Photon Source, Argonne National Laboratory. An energy of 15 kev corresponding to a wavelength λ = 0.83 Å was selected using a double-crystal monochromator. The data were collected using a CCD detector (MAR) positioned 245 cm behind the sample. The scattering intensity was recorded in the interval 0.005<q<0.23 Å -1. The wave vector defined as q = (4π/λ)sin(θ/2), where θ is the scattering angle. Samples of PA 1 with each of PAs 1a-1e were lyophilized from 10% NH 4 OH (1,1,1,3,3,3- hexafluoro-2-propanol), resuspended in NaCl (100 mm) at 1 wt%, and analyzed in 2 mm quartz capillaries. The two-dimensional SAXS images were azimuthally averaged to produce one-dimensional profiles of intensity (I) vs. q, using the two-dimensional data reduction program FIT2D. Scattering of a capillary containing only solvent was also collected and subtracted from the corresponding data. No attempt was made to convert the data to an absolute scale. Figure S3. (a) SAXS of composite nanofibers of 90% PA 1 and 10% spin labeled PAs 1a-1e show nearly identical scattering profiles, confirming that the incorporation of PAs 1a-1e does not disrupt the supramolecular structure even at high (10%) loading of spin labeled PA. The scattering profiles indicate the presence of high-aspect-ratio nanofibers with diameters comparable to those measured by TEM image analysis. (b) SAXS of PA 1 nanofibers, composite nanofibers of PA 1/2, and PA 2 alone. Scattering profiles show that the PA 1 nanofiber geometry is unaffected after incorporation of PA 2 at 20%. When PA 2 is incorporated at 50% the -1 slope at low q shows that a high-aspect-ratio geometry still exists with only slightly changes in the geometry. SAXS of PA 2 alone, in contrast, does not show assembly into high-aspect-ratio nanofibers. 3. Dynamics characterization by EPR 4 NATURE MATERIALS

5 3.1 Anisotropic diffusion model. To reflect the anisotropic motion expected for radical electron spin labels covalently bound to peptide amphiphiles, a single dynamics parameter, rbar, was used together with the anisotropy parameter, n. With this method, we obtained good approximations for dynamics as previously established for other peptides spin labeled with TOAC. 3 Each site through the PA nanofiber cross-section was characterized by rbar (where 10 rbar = k r ) and n, (where n = rprp/rpll, where rprp and rpll are the rotational diffusion coefficients perpendicular and parallel to the magnetic field vector, respectively). Table S1 shows that the anisotropy parameter, n, deviates further from 1 in regions of the nanofiber that are expected to exhibit greater anisotropy. 3.2 EPR fitting procedure and statistical treatment. All g and a tensor values were determined from the spectra of each PA at 150 K and were fixed through all the fitting procedures. Best fits of spectra at the rigid limit were obtained when ax=ay, so the constraint, ax=ay=aprp was imposed, and fitting of the a tensor was done with parameters, aprp and apll. Imposing this constraint on room temperature spectra also yielded best fits. The best fits are shown in Fig. S4 with the output parameters summarized in Table S1. The rotational diffusion rates (k r ) calculated from the rbar values in Table S1 are presented in Fig. 3a. The error bars in Fig. 3a correspond to the range of output rbar values that returned acceptable fits from systematically varying the input seed values. The error bars represent the deviation from the global best fit, and each was determined from the output rbar values of a set of 40 simulations. Figure S4. EPR spectra (black lines) and their best fits (red lines) obtained by fitting with the NLSL program (a) PA 1 nanofibers with 0.4% of each PA 1a (top) through 1e (bottom), and (b) PA 1/2 nanofibers with 0.4% of each PA 1a-1e; Sweep widths: 150 G. Table S1 Output parameters from EPR global fits. Each sample contains one spin labeled PA mixed into PA 1 of PA 1/2 composite nanofibers at 0.4%. All spectra of PA nanofibers were simulated with 1- component fit with the exception of PA 1/1a, 1/2/1a, and 1/2/1c. In these cases, the output parameters reported are those of the dominant components (containing >90% of each sample). The G tensor values were determined from frozen (100K) spectra and were fixed in each fit at {gx, gy, gz}={2.0095, , }. NATURE MATERIALS 5

6 PA 1 Radial Nanofibers position rbar n c20 wx wy wz aprp apll PA 1/1a* PA 1/1b PA 1/1c PA 1/1d PA 1/1e PA 1/2 Radial Nanofibers position rbar n c20 wx wy wz aprp apll PA 1/2/1a* PA 1/2/1b PA 1/2/1c* PA 1/2/1d PA 1/2/1e * Output parameters reflect the major component of a 2-component fit 3.3 The presence of two components in spectral simulations. In Fig. 2, the spectra corresponding to PA 1/2 with spin labels 1c and, to a small extent, 1a, exhibit two components. These spectra were fit with two component simulations in the NLSL program. In these cases, integration of the fits shows the dominant (broad) component to comprise > 98% of the constituent spin probes. The minor component reveals faster motion, approaching to that of a freely dissolved spin label, and is likely due to a small concentration of monomeric or micellar PA 1a or 1c in solution. While the percent spin label outside of a nanofiber is very low, this effect is more significant when the spin label is positioned at a site that is likely to disrupt the packing of PAs, as in a nanofiber that has weaker intermolecular cohesion (as in PA 1/2 that contains no β-sheet character). 1 Teixidó, M., Albericio, F. & Giralt, E. Solid phase synthesis and characterization of N methyl rich peptides. The Journal of peptide research 65, (2005). 2 Karim, C. B., Zhang, Z. & Thomas, D. D. Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes. Nature protocols 2, (2007). 3 Karim, C. B., Kirby, T. L., Zhang, Z., Nesmelov, Y. & Thomas, D. D. Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone. Proc. Natl. Acad. Sci. USA 101, (2004). 6 NATURE MATERIALS