# Peptide Mediated Antimicrobial Dental Adhesive System
## 3 Results and Discussion
### Computational Structure Flexibility  
Secondary structures of peptide sequences can also be computationally predicted by generating structure decoys through PyRosetta. The distribution of predicted secondary structural features as a function of sequence residue for GH12 and GH12-M2 are shown in Figure 4a,b. Darker colors denote higher frequencies of the feature labeled at the right-hand side. The residue positions with the highest occurrence of alpha helix are residues 3-7 (LWHLL). The residues with the highest predicted three-helix structure frequencies are residues 3-5 (LWH). It should be noted that the first residue and last two residues cannot be labeled with these three-helix structure features because the definition used by DSSP (Define Secondary Structure of Proteins) requires the presence of adjacent residues. Bends (which are not helices) peaked at residues 5, 9, and 10. Turns, not defined as bends or helices, peaked at residues 2 and 3. The bottom two rows are defined by the direction in which the peptide backbone rotates. For about 80% of the decoys, the leucine at residue 2 is turned right, from residue 3 to 6 about 70% of decoys are turned right, and for residues 7-10 about 60% of the decoys. Figure 4c shows the helix in the GH12 peptide backbone ribbon for the lowest energy score decoy. Figure 4d represents the GH12-M2 peptide backbone ribbon structure for the decoy with the lowest calculated energy score. No \(\alpha\)-helical secondary structure was predicted for this decoy. However, the sequence modification of M2 (GSGGGK) did induce a change in the flexibility analysis results across residues 2-10 as compared to the same residues of the GH12 decoy. Even though no helix features were predicted for the lowest energy score decoy, 3\({}_{10}\)-helix structures were observed for about 10-20% of decoys across residues 2-7. The percentages of \(\alpha\)-helix dropped below 10% for all residues \begin{table}
\begin{tabular}{c c c} \hline \hline
**Shift Peak Structure** & **Shift Peak (cm\({}^{-1}\))** & **Peptides** \\ \hline \(\alpha\)-Helical structures & 1617, 1652 & GH12 (ratio: 0.6), GH12-M2 (ratio: 0.5) \\ CH2 CH3 deformations & 1428 & GH12, GH12-M2 \\ \(\beta\)-Strand conformation & 1259 & GH12, GH12-M2 \\ Unordered/turn & 856, 828 & GH12, GH12-M2 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Raman spectra of GH12 and GH12-M2 by Diode785. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Peak Region Name** & **Peak (cm\({}^{-1}\))** & **Peptides** \\ \hline Amide I & 1658, 1652 (\(\alpha\)-helical structures) & GH12 (twin), GH12-M2 \\ Amide II & 1536 & GH12, GH12-M2 \\ Amide III & 1338 (\(\alpha\)-Helical structures) & GH12, GH12-M2 \\ Amide III & 1259 (Unordered/turn) & GH12, GH12-M2 \\ CH3,N-H,C-N & 1027 & GH12-M2 \\ stretching-bending [83] & 1027 & GH12-M2 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: FTIR spectroscopy of GH12 and GH12-M2. of GH12-M2 compared to \(\alpha\)-helix structure predictions above 10% for residues 3-7 for GH12. The GH12-M2 flexibility features of bend were calculated in residues 14 and 16 in about 50% of the decoys. The right and left turn frequencies were similar to those observed for GH12, with a 50-50% split for the spacer residues. Although the structural analysis of CD for both GH12 and our modified derivative peptide, GH12-M2, showed significant differences, FT-IR and Raman spectra analyses of both GH12 and GH12-M2 displayed only slight variations. The difference in structure flexibility between GH12 and GH12-M2 appeared negligible in accordance with the fragment insertion method employed by PyRosetta. The decoy generation method is stochastic and therefore cannot accurately predict flexibility changes if the lowest energy structures possible are found. However, the lowest energy structure may differ from the native structure [84; 85]. The parallel optimization of many decoys at once may also provide insight into the distribution of the ensemble of structures sampled by peptides in solution.