De novo designed peptides form a highly catalytic ordered nanoarchitecture on a graphite surface

Here we demonstrate that short peptides, de novo designed from first principles, self-assemble on the surface of graphite to produce a highly robust and catalytic nanoarchitecture, which promotes peroxidation reactions with activities that rival those of natural enzymes in both single and multi-substrate reactions. These designable peptides recapitulate the symmetry of the underlying graphite surface and act as molecular scaffolds to immobilize hemin molecules on the electrode in a hierarchical self-assembly manner. The highly ordered and uniform hybrid graphite–peptide–hemin nanoarchitecture shows the highest faradaic efficiency of any hybrid electrode reported. Given the explosive growth of the types of chemical reactions promoted by self-assembled peptide materials, this new approach to creating complex electrocatalytic assemblies will yield highly efficient and practically applicable electrocatalysts.


Peptide self-assembled structures observed by in -situ AFM.
Figure S1. In-situ AFM height images of peptide assemblies formed by 1 M aqueous solution of peptide 1.
In this manuscript, AFM images measured under wet conditions were named "in-situ AFM image", while AFM images measured under dry conditions were named "ex-situ AFM image".
In-situ AFM was utilized to observe the self-assembled structure at a high resolution (Fig. S1).
The in-situ AFM (Cypher, Asylum Research, Oxford Instruments) was performed under DI water.
Before the measurement, the graphite surface was incubated with 1-M solution of peptide 1 for 1 hour, and the peptide solution was replaced with DI water. The AFM cantilever for the in-situ AFM was BL-AC40TS-C2, Biolever mini, Olympus. The high-resolution AFM image shows clear nanowire structures of peptide 1, which are uniformly assembled on a graphite surface with a periodicity distance of 5 nm (Fig. S1). The length of the peptides with 7 amino acids in this work is 2.5 nm. The 5-nm distance corresponds to the length of two peptides. These uniform structures can be observed in in-situ AFM imaging, but it is not possible in the case of ex-situ AFM measurement. The self-assembled structures of peptides can be denatured during the drying process. It is worth noting that the coverage and domain size of the self-assembled structures can be maintained even after the drying process.    showing that the height increased drastically after 30 mins.

Peptide self-assembled structures observed by ex-situ AFM.
The concentration dependence of the peptide self-assembly was examined by ex-situ AFM. The Ex-situ AFM images were taken after drying the samples. The AFM images have a size of 2 x 2 m 2 to observe the uniformness and coverage of the structures. As shown in Fig. S4, these peptides

Estimation of peptide height and coverage from ex-situ AFM images.
To estimate the peptide coverage, we converted the height images ( Fig. S4) into height histograms ( Fig. S5) and fitted them with Gaussian functions to identify peaks. The peak at lower height was assigned to the bare surface of graphite, while the peaks at higher height were attributed to the peptide self-assembled layer. The peak position indicates the thickness of the peptide layer, varying in the range of 0.5-1.2 nm depending on the concentrations and the kind of peptides for the self-assembly (Fig. S5). Utilizing the area of each peak, we estimated the coverage of peptides on the surface. 1 We found that peptide 4 (VHVHVYV) showed three peaks attributed to the bare graphite surface and two phases of peptide assembled structures on the graphite surface. Each peptide phase has a height of 0.5 and 1.0 nm, respectively. The doubled thickness of another phase indicates that the phase with the 1.0-nm thickness can be a bilayer of the phase of 0.5-nm thickness. Interestingly, the bilayer formation of peptide 4 can be correlated to multiple valines in the sequence, whereas the other peptides with multiple leucines form monolayer-like assemblies.
As increasing the concentration of peptide solution, the coverage of peptides increases. The coverages of peptides versus concentrations were plotted and fitted with the Langmuir isotherm model to estimate the peptide binding affinity to graphite (Fig. S6). The dissociation constants K d of 1, 2, 3, and 4 were estimated to be 294, 125, 208, 275 nM, respectively. The graphite surface was fully saturated at peptide concentrations of more than 1 M. Thus, we utilized this concentration for the rest of the experiments.  The stability of the self-assembled structure of peptides was characterized by the following method. After one-hour incubation of peptide solutions on a graphite surface, the self-assembled structures were immediately washed with 100 L of Milli-Q water. We replaced the incubation solution with fresh Milli-Q water every 30 seconds and repeated the washing process five times.
After the fifth wash, the excess water was blown off by nitrogen gas, and the samples were dried overnight. Then, morphologies of the self-assembled structures on graphite were observed by exsitu AFM. The coverage of peptides on graphite did not change after the washing process, proving the outstanding stability of the self-assembled structures under Milli-Q water. The ordered structures formed by the peptides were also unaffected by washing with H 2 O 2 (Fig. S8). In the H 2 O 2 case, we duplicated the above washing process except for using H 2 O 2 instead of Milli-Q water. drastically increased after 30 minutes, implying the aggregation or standing-up of hemins on the self-assembled peptides (Fig. S3b).

Calculation of hemin density on the self-assembled structure.
The coverages of hemin on the self-assembled structure were calculated by analyzing the area of the higher peak of each plot (Fig. 2).       The electron transfer coefficient  and the equilibrium potential V t were estimated using the Butler-Volmer equation. (4), where is the electrode current density, A/m 2 ; is the exchange current density, A/m 2 ; is the 0 number of electrons involved in the electrode reaction; is the Faraday constant; is the gas constant; is the absolute temperature (K); is the electrode potential, V. The cyclic voltammetry measurements of peptides-hemin hybrid electrodes were carried out at different scan rates (0.1-1 V/s) in the phosphate buffer (0.1 mol/L, pH = 7.0). The heterogeneous electron transfer rates (K s ) was estimated from the peak difference (∆E p ) and the scan rate (v) by using Laviron's approach, where α is the electron transfer coefficient; n is the number of electrons transferred in reaction; R is the gas constant; T is the absolute temperature. ∆E p was derived from the peak positions in the cyclic voltammograms with various scan rates. Based on the fitting with equation 5 (Fig. S14), the values of ∆E p and K s for different scan rates were given in Table S3 and S4, respectively.