## 3 Results
### Potential Binding Sites for Collagen Type I Alpha Chain 2  
The high-stability collagen-binding peptide's predicted interactions covered more of the pH range than with TKKTLRT alone. To see this trend, we investigated the screening method's binding interactions across all of the residues of COL1A2. This analysis was a first approximation that did not include the hydropathy change from proline to hydroxyproline, which is more electronegative with the addition of the hydroxyl group. Because proline is an electropositive amino acid before the addition, the hydropathy is closer to octanol than to water after the substitution. Hydrophobic interactions are more likely than they appear in our screening method. See Figure 6 for the likelihood of binding for the TKKTLRT (magenta) and SQNPVQP (gold) peptides across the COL1A2 residues. The pH independence of the high-stability collagen-binding peptides comes from its ability to interact more readily with hydrophobic interfaces like LRR-10 (See Figure 7). The inserted leucine gives the designed peptide a binding site with similar potential binding free energy to LRR-10, which is achieved without depending on the pH state of any of its residues. We had some predicted binding sites for TKKTLRT at pH 2, and we had no predicted binding sites for TKKTLRT at pH 2. The TKKTLRT still retains the strong predicted binding with COL1A2 sub-sequences with D and/or E amino acids, which are up to 1 kcal/mol more favorable than the LRR-10 hydrophobic binding sites. The increase in potential energy of binding comes from the difference in hydrophobic character of protonated histidine from the LRR-10 interface and hydrophobic character of replacing histidine with threonine from the peptide analogue interface at pH 2. This difference implies that the binding sites between COL1A2 and the peptide analogue are not likely to be reversed, whereas the LRR-10 binding sites are likely to be reversible as histidine changes protonation states. At more alkaline pH values, both peptides have binding sites which are not likely reversible. The exception is that LRR-10 binding sites are reversible by protonation of its histidine. The trends of Figure 7 interactions may also apply to other extracellular matrix proteins. The potential binding sites of these peptides were evaluated for decorin, the protein origin of LRR-10, in Figures S3 and S4. First, we showed that collagenase binding peptide does not have strong electrostatic interactions with decorin, which is similar to COL1A2. The peptide sequence lacks charged residues, so strong electrostatic interactions with the collagenase peptide are lacking. For TKKTLRT and TKKTLRT, electrostatic interactions are possible. We also highlighted an important characteristic of COL1A2, which is that the triple helix structure increases the availability of residues compared to other protein tertiary or quaternary structures. The LTL motif had many predicted hydrophobic interactions with decorin. However, due to energy landscape minimization constraints, hydrophobic interactions are less likely to be available depending on the strength of hydrophobic interactions. Therefore, the interacting partners for these interactions are likely to be interior for most proteins. However, if proteins are unfolded, then these interactions may become available. Similar to the LTL motif in the high-stability collagen-binding peptide, the LHL motif in LRR-10 was predicted to have many strong hydrophobic interactions with decorin if those sites are available. Figure 6: Free energy of binding estimates for sets of three adjacent residues for three different collagen-binding peptides for all residues of human collagen type I alpha chain 2 (COL1A2). Positive binding sites are energetically unfavorable, while negative binding sites are favorable. Binding sites significantly below \(-\)kT are likely to be stable. Each circle represents a potential three-amino-acid binding site between a peptide and collagen.