Peptide synthesis and purification

Peptides were synthesized by CEM Discover microwave synthesizer using Fmoc chemistry at 100-μmol scales. The Fmoc protecting group was removed by piperidine/dimethylformamide solution (20/80 v/v); at each coupling step reactants were added with the amino acid:HBTU:DIEA:resin ratio of 5:4.9:10:1. Products were cleaved from the H-Rink Amide-ChemMatrix (PCAS, 0.53 mmol g−1 loading) in a cleavage cocktail solution (trifluoroacetic acid (TFA)/triisopropyl silane/deionized water, 95/2.5/2.5 v/v) for 2 h and the remaining solution was vapourized with N 2 gas. Peptide was precipitated with cold diethyl ether (Aldrich) and dried in vacuum. After dissolving the peptides in DI H2O, purification proceeded by preparative reverse-phase high-performance liquid chromatography (Waters prep 150 LC System) using preparative C4 column (XBridge BEH300 Prep C4 5 um) and a linear gradient of buffer A (99.9% H2O and 0.1 % TFA) and buffer B (90% acetonitrile, 9.9% H2O and 0.1 % TFA). Molecular mass of the peptide was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Bruker Ultraflex III). Products had over 95 % purity.

Preparation of peptide/fullerene solutions

Samples were prepared with 8 mg ml−1 protein solution (COP) in 25 mM Tris pH 8.0 buffer solution and 1 mg C 60 or C 60 pyrrolidine Tris-acid (Aldrich). Fullerene powder was mixed with pre-made 0.2 ml of 8 mg ml−1 protein solution in 25-mM Tris pH 8.0. The sample was then tip-sonicated (QSonica, Q125, 1/8th inch tip) on an ice bath for 5 min to be saturated of fullerene. Ice-bath cooling was to prevent excessive sample heating and destabilization of protein structure. The sonicated samples were warmed up to room temperature and centrifuged at 14,500g for 10 min (Eppendorf, Centrifuge 5430R).

Ultraviolet–visible absorption spectroscopy

Ultraviolet absorption spectra of the COP alone and C 60 /COP were recorded using a Hewlett Packard 8453 spectrometer in 1 cm Hellma Quartz SUPRASIL (QS) cells. The COP and C 60 /COP were prepared in a buffer of 20 mM sodium phosphate, 100 mM NaCl and pH 7.5. Ultraviolet–visible spectra of C 60 /COP and COP were used to roughly estimate the concentration of solubilized fullerene by absorbance at 340 nm (the molar absorptivity of 49,000 M−1 cm−1 was used for C 60 , (ref. 28). The resulting molar concentration of solubilized C 60 in the C 60 /COP solution was 6.22 μM (compared with COP at 585 μM in the same solution).

Size-exclusion chromatography

Size-exclusive gel filtration elution profiles were obtained using a Superdex 75 10/300 GL column with a GE Healthcare fast performance liquid chromatography (FPLC) system (Amersham Pharmacia Biosystems). Peptides (at 200 μM) were prepared in a buffer of 20 mM sodium phosphate, 100 mM NaCl and pH 7.5 at room temperature. 200 μl of each sample were loaded and eluted with the same buffer. The column was equilibrated in 20 mM sodium phosphate, 100 mM NaCl and pH 7.5 with a mobile phase flow rate of 0.5 ml min−1, and absorbance at 220, 280 and 340 nm was recorded. Calibration curves were obtained using the molecular-weight standard kit, MWGF70 6,500–66,000 (Supplementary Fig. 10).

Analytical ultracentrifugation

Oligomerization states of COP and C 60 Sol–COP were determined by equilibrium sedimentation performed at 25 °C using a Beckman XL-I analytical ultracentrifuge. Both solutions were prepared in a buffer of 25 mM Tris pH 8.0. Equilibrium radial concentration gradients at four different rotor speeds (25, 30, 35 and 40 K r.p.m.) were acquired as absorbance scans at 340 nm for C 60 Sol with COP and 280 nm for COP peptide alone. Data were globally fit to single-species or two-species models of equilibrium sedimentation by a nonlinear least-squares method using IGOR Pro (Wavemetrics), and the best-fitting model was accepted38. Supplementary Figure 4 shows sedimentation equilibrium profiles of C 60 Sol–COP along with corresponding species distribution plots consistent with a tetramer–octamer equilibrium, whereas COP alone appears as a tight tetramer. This is consistent with results from SEC, shown in Fig. 1c and Supplementary Fig. 2.

Crystallization, data collection and processing

The first X-ray diffraction quality crystal (C 60 Sol–COP-1) was obtained by the hanging-drop vapour diffusion technique at 291 K, over a period of 15 days in a 2 μl drop consisting of 1:1 v/v mixture of 1 mgml−1 protein solution in 20 mM sodium phosphate/100 mM NaCl pH 7.5 buffer and a reservoir solution of 17 mM lithium sulfate monohydrate, 85 mM Tris-hydrochloride sodium pH 8.5, 25.5% polyethylene glycol (PEG) 4,000, 25% v/v glycerol (Hampton Research sparse matrix). The crystal was flash-frozen, and diffraction data were collected at the 24-ID-E NE-CAT beamline at the Argonne National Laboratory. Data sets were indexed and integrated with MOSFLM39,40, and scaled using SCALA3 (Collaborative Computational Project, Number 4, 1994)41. Diffraction data were recorded to a maximum resolution of 2.35 Å (Table 1).

Subsequent crystallization attempts were performed with higher concentrations of the C 60 Sol/COP suspension, using commercially available sparse-matrix screens from Hampton Research and the hanging-drop vapour diffusion method at 295 K. Diffraction-quality crystals of C 60 Sol–COP (C 60 Sol–COP-3) were obtained by mixing equal volumes of the C 60 Sol–COP mixture at 8 mg ml−1 in 25 mM Tris pH 8.0 and reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, 30% w/v polyethylene glycol 4,000. Microcrystals grew within 24 h, with larger oval-shaped crystals appearing in several days (Supplementary Fig. 5). Crystals were cryoprotected using reservoir solution supplemented with an additional 30% (v/v) glycerol and were flash-cooled in liquid nitrogen. Diffraction data, extending to 1.67 Å resolution, were collected at 100 K on beamline 7A equipped with an ADSC Quantum 270 CCD detector at Pohang Accelerator Laboratory (PAL, Pohang, Korea). The C 60 Sol–COP complex crystal belonged to space group P6 2 , with unit cell parameters a=b=42.1, c=66.7 Å, α=β=90.0 and γ=120.0°. Data were processed and scaled using the programs DENZO and SCALEPACK from the HKL-2000 program suite42. The Matthews coefficient43 for C 60 Sol–COP was 2.54 Å3 Da−1 and the estimated solvent content was 51.5%; there were two COP molecules and one C 60 Sol in an asymmetric unit.

In addition to the above, diffraction-quality crystals were also obtained in three other conditions (1.5 M ammonium sulfate, 0.1 M Tris pH 8.5, 12% v/v glycerol; 0.1 M HEPES–Na pH 7.5, 0.8 M potassium sodium tartrate tetrahydrate; and 0.1 M N-(2-acetamido)iminodiacetic acid, N-(carbamoylmethyl)iminodiacetic acid (ADA) buffer pH 6.5, 1 M ammonium phosphate dibasic), in each case yielding identical unit cell and space group, thus showing the same assembly geometry. Crystals grown under the latter condition diffracted to 1.76 Å at a home source (C 60 Sol–COP-2).

Structure solution and refinement

For all the data sets, structure determination was carried out by molecular replacement using the programme PHASER44. The Matthews coefficient suggested a dimeric helix in the asymmetric unit. Molecular replacement calculations were performed using the dimeric unit of a polyalanine model obtained from coordinates of previously solved crystal structure 3S0R as the search probe. The solution model was subjected for rigid body refinement followed by iterative model building and restrained refinement protocols implemented in Auto Build module of PHENIX45. All side chains were traced in the electron density map. During the course of map tracing, electron density for fullerene was clearly visible and modelled for refinement.

During data analysis, it was found that the crystal (C 60 Sol–COP-1) was merohedrally twinned. The H-test results, |H|=0.024 (0.50 for untwinned and 0.0 for 50% twinned) and H2=0.001 (0.33 for untwinned and 0.0 for 50% twinned), were indicative of merohedral twinning with the twin law (h, -h-k, -l), where H=|I 1 −I 2 |/|I 1 +I 2 |, I 1 and I 2 are twin-related acentric reflections. The cumulative distribution of H46,47 and Britton plots48,49 estimated twin domain fraction (α) to be 0.478 and 0.447, respectively.

As the estimated twin fraction was close to 0.5, the data were not detwinned for further refinement. Instead, the refinement was carried out by refining both parameters of the model and twin fraction. The PHENIX45 refinement protocol, which implements this option, was used.

The PHENIX refinement protocol was used. Upon converging, the refinement strategy produced model with good R work /R free statistics in each case (Table 1). Model building was further carried out manually using COOT50. Structure figures were created using the programme PyMOL (Schrödinger, LLC). Crystallographic data statistics are summarized in Table 1.

Coiled-coil parameter fitting

Parameter fits were performed using CCCP ( http://grigoryanlab.org/cccp) via the ‘global symmetric’ fit option, where ideal symmetry (in this case D2) is assumed30. The apo and C 60 -bound structures fit within 0.6 and 0.4 Å, respectively, indicating that they both closely resembled an ideal coiled coil. Key parameters are listed in Supplementary Table 1. Detailed parameter definitions and information on the fitting procedure can be found in reference30.

Binding free-energy calculation

The NAMD 2.10 software package, developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign51, in conjunction with CHARMM22 force field52 was used for this study. A new atom type was created for the C 60 carbon (CA60), which was identical to the aromatic carbon atom type in CHARMM22 (type CA) in all aspects except for the equilibrium CA60–CA60 bond length, which was set to 1.4392 Å to match the experimentally observed average bond length in C 60 (ref. 53). All simulations were performed in explicit TIP3P water; a padding of 8 Å was used for initial solvation, with sodium/chloride counterions added to achieve charge neutrality as necessary. Periodic boundary conditions were applied and all simulations were performed in the NTP ensemble at 298.15 K and 1 atm. Explicit calculation of long-range interactions was cutoff at 10 Å, with a switching function starting at 6 Å. Particle Mesh Ewald method was used to correct for long-range electrostatics54 and an analytical correction was used to capture long-range van der Waals interactions55. Pande and co-workers have shown that with these corrections, the 6/10 Å non-bond cutoff schedule performed as well as longer cutoffs in free energy of solvation calculations56.

To compute the free energy of C 60 –COP association, we followed the double-decoupling framework outlined by McCammon and co-workers31. In this approach, one seeks to compute the free energy of decoupling the ligand (here C 60 ) from the rest of the system when it is either bound to the receptor (here COP) or solvated by itself. The standard-state free energy of binding is then related to the difference between the two decoupling free energies, appropriately corrected for the standard-state concentration31. We sought to use the method of FEP to compute individual decoupling free energies, but the direct application of the method to C 60 exhibited very strong hysteresis between forward and reverse simulations (that is, decoupling C 60 and coupling it back, respectively). Because C 60 is hollow, with enough space inside for several water molecules, as the molecule is decoupled, water rushes in to occupy the available space. However, during the reverse simulation, as C 60 is coupled back to the system, water molecules tend to remain trapped inside the fullerene, leading to a very different end state. Note that use of the soft-core van der Waals scaling55, implemented in NAMD, does not resolve this issue as there is little to encourage water molecules to escape the core of fullerene as it is coupled back. This very large hysteresis meant that we could not claim good convergence (and hence accuracy) of either forward or reverse simulations.

To resolve this issue, we introduced an intermediate step in the C 60 decoupling/coupling transformation, designed to provide reversibility, slightly adjusting the double-decoupling framework. The idea was to introduce an artificial atom, with a size to roughly match the radius of C 60 , which could be used to ‘make room’ for C 60 before the molecule is coupled to the system. A new atom type was created, called C60D (for C 60 ‘dummy’), with a van der Waals radius of 4.5 Å and a Lennard-Jones well depth of −1.0 kcal mol−1. Because C60D is a single atom, and not hollow-like C 60 , the soft-core van der Waals potential will indeed gradually repel water molecules as C60D appears. Thus, the two decoupling transformations were altered as follows:

where subscripts wat and gas indicate that the corresponding molecule is either fully coupled to the system (that is, in water) or fully decoupled from it (that is, in the gas phase), respectively. The initial state of transformation 1 involves the complex between COP and C 60 ( ) and a decoupled C60D ( ) overlapping the fullerene. The first step of the transformation involves decoupling C 60 from the system, while C60D is coupled, such that the intermediate step has C 60 in the gas phase (C60D gas ) while C60D is fully interacting with the system, occupying the fullerene-binding site ( ). The second step of the transformation then decouples C60D as well, such that the end state involves COP in solvent alone with both C 60 and C60D in the gas phase. Because gaseous C60D is present in both end states, its contribution to the total free-energy difference cancels, such that the net transformation still represents just the decoupling of C 60 . On the other hand, the presence of C60D and the intermediate state address the reversibility of the transformation. Because the first step in the reverse direction involves coupling of C60D, room is created in the solvent before C 60 is reintroduced and C60D is once again decoupled in the second step. To prevent C60D from diffusing away from the binding site at any point in the simulation, harmonic restraints were applied between C60D, and Cγ, Cɛ 1 and Cɛ 2 atoms of the binding site Tyr (residue 9), with equilibrium distances of 6.7, 6.7 and 7.0 Å, respectively (taken from the crystal structure by initially placing C60D in the geometric centre of the bound C 60 ), and a force constant of 10 kcal mol−1 Å−2. Note that these restraints do not contribute to the FEP calculation (since their energy is independent of the coupling parameter) and their presence fully cancels between end states of the transformation. Another restraint was needed to make sure C 60 does not diffuse far from the binding site when decoupled, which would create convergence difficulties. A harmonic restraint was applied between the centre of mass of C 60 and C60D, with an equilibrium distance of zero and a force constant that increased from 0 to 10 kcal mol−1 Å−2 as C 60 was decoupled from the system. Specifically, if λ is the FEP coupling parameter for the current window (with 0 and 1 corresponding to C 60 being fully coupled and decoupled, respectively), the force constant used was kcal mol−1 Å−2. The energy of this restraint was accounted for in FEP calculations, so that the final free-energy change for transformation 1 represented the difference between a state where C 60 is fully coupled and bound to COP and one where C 60 is decoupled from the system, but harmonically restrained to remain in the vicinity of the binding site. To remove the influence of this restraint and correct for the standard state, this free-energy change was corrected by , where is the standard-state concentration and k is the force constant of the C 60 restraint in the decoupled state (that is, 10 kcal mol−1 Å−2)31,57.

Transformation 2 is similar to transformation 1, but with no protein. In the first step, C 60 is decoupled from solvent as C60D is coupled, whereas the second step decouples C60D. As with transformation 1, the influence of C60D cancels between the two end states, with the total free-energy difference corresponding to that of decoupling C 60 from solvent. However, the intermediate step again renders the path reversible. As with transformation 1, here it was important for C 60 and C60D to be approximately coincident throughout the simulation (so that, for example, in the first step of the reverse simulation coupling of C60D creates a cavity in the right location within the solvent for C 60 to couple into later). For this reason, a harmonic restraint was introduced between the centroid of C 60 and C60D, with equilibrium distance of zero and a force constant of 10 kcal mol−1 Å−2. Note that the contribution of this constant restraint cancels between the two end states (so the total free-energy change of transformation 2 is still that of decoupling C 60 alone), and its energy does not influence FEP calculations.

Since C 60 remains decoupled (and restrained to C60D) throughout step 2 of both transformations, it does not contribute to the free-energy change associated with these steps. For this reason, C 60 need not be explicitly present in simulations of these steps and was omitted for simplicity.

FEP details and results. NAMD’s alchemical transformation module (in conjunction with the FEP method) and the collective variable module (for introduction of restraints) were used to implement the above transformations. The soft-core van der Waals radius-shifting coefficient (parameter alchVdwShiftCoeff) was set to 8 Å2 in the first step of both transformations and to 20 Å2 in the second step of both transformations (values were chosen to produce smooth transitions in short FEP test runs). All four steps were carried out using 20 FEP windows, with the coupling parameter varying uniformly from 0 to 1. Each window involved 10 ps of equilibration followed by 190 ps of data collection. At the start of each simulation, the system (upon being minimized for 1,000 steps) was pre-equilibrated for 200 ps. Each step of both transformations was run 10 times in both forward and reverse directions, using different random seeds. Thus, a total of 336 ns of simulation was performed. The final results are summarized in Supplementary Fig. 8a, where values for reverse transformations have been negated to represent free energies in the decoupling direction. Error bars represent s.e.’s of the cumulative free-energy difference, computed over the 10 simulations run for each step/direction combination. Clearly, all steps exhibit excellent convergence and reversibility. The standard-state free energy of C60–COP binding was computed as:

where is the free-energy change of the ith step of transformation K. The final estimate amounted to −9.8±0.3 kcal mol−1, where the uncertainty was calculated by error propagation using s.e.’s emergent from combining all simulations of each step (both forward and reverse).

Association of fullerene with individual aromatic groups

An analogous approach was also used to calculate the affinity of C 60 for a disembodied Tyr residue (acetylated and methyl-amidated on the N- and C termini, respectively) and a Tyr side-chain analogue (p-methylphenol). The only difference was that in these cases an additional constant harmonic restraint, between the centre of mass of C 60 and C60D, was added throughout step 1 of transformation 1. This restraint, with a force constant of 1.0 kcal mol−1 Å−2 and equilibrium distance of 0 Å, prevented C 60 from dissociating from the bound molecule in the initial FEP window, which otherwise occasionally occurred in some trajectories and limited the amount of useful sampling. The effect of this restraint was removed from the final estimate by applying the standard importance sampling formula58 to adjust the expectation computed in FEP59. The final standard-state binding free-energy estimates were −1.76±0.15 for C 60 and isolated Tyr, and −1.53±0.07 for C 60 and p-methylphenol (Supplementary Fig. 8b–c). These correspond to dissociation constants in the mM range, meaning that the affinity is expected to be extremely weak.

Designability analysis

To estimate the natural abundance of structural motifs surrounding the C 60 -binding site, search engine MASTER33 (grigoryanlab.org/master) was used to search a highly non-redundant subset of the PDB. Specifically, the weekly BLASTclust-based clustering60 of all PDB chains was downloaded on 22 October 2014, and the first chain from each cluster selected, filtering for X-ray structures resolved to 3 Å or below. The asymmetric unit of each of the entries was then downloaded and the crystallographic lattice generated, keeping all images that were reasonably close to the initial unit (defined as having at least three atoms within 16 Å of any atom in the initial unit). The resulting lattices were then combined into a MASTER database of 13,400 entries. All searches were performed using the full-backbone setting of MASTER, which provably finds the closest matches to the query in terms of the heavy-atom backbone r.m.s.d. (that is, N, CA, C and O). The full C 60 -binding motif was defined as residues 2–9 on one pair of chains and 19–24 on the opposing pair, with individual interfaces of this motif defined accordingly (Supplementary Fig. 7b). Sequence logos in Supplementary Fig. 7b were generated by considering all matches within 0.3 Å and discarding those with identical sequences (although the database is highly non-redundant, matches of identical sequence are still possible when multiple-matching instances are found within the same lattice).

Measurement of electrical conductance

Current versus voltage curves were obtained using the variable temperature microprobe system from MMR technologies coupled with HP 4145B semiconductor parameter analyser. The samples were deposited on a degenerately doped silicon substrate with 200The thermal oxide, which was photolithographically pre-patterned with Au/Cr (45 nm/5 nm) electrodes. The channel length and width were 10 and 6,000 μm, respectively.