# Peptide Mediated Antimicrobial Dental Adhesive System
## 3 Results and Discussion
### Effect of Peptide Conformation on Function  
Structure-function relations are critical in peptide function, hence we explored the structural differences that may occur between modified GH12-M2 and unmodified GH12 prior to studies on peptide coupling to adhesive formulation. First, we performed circular dichroism spectroscopy to investigate the secondary structure and folding properties of the peptides in solution. We used different buffers to test the conformation of the peptides including a range of concentrations of trifluoroethanol (TFE) to mimic their folding behavior at the solid interfaces. The circular dichroism spectrum was relatively flat for GH12 without TFE in ammonium sulfate and potassium phosphate buffers at pH 7.4 with a negative peak near 230 nm as shown in Figure 2. The CD spectrum for GH12-M2 in buffer only showed a similar trend to GH12, with a negative peak near 235 nm. In the presence of 10% TFE, spectral flattening still occurred for both peptides with a slight increase in the MRE near 185 nm. Increasing the TFE to 20% resulted in a large difference in the CD spectrum among the peptides. Specifically, the GH12-M2 peptide in the 20% TFE solution demonstrated spectral features characteristic to the helical formation of secondary structure, including a large, positive peak near 190 nm and negative peaks near 205 and 225 nm. In contrast, the GH12 peptide alone did not show this behavior in the same 20% TFE solution. However, TFE concentrations of 40% and above elicited spectra characteristic to helical features for both the GH12 and GH12-M2 peptides.  
Detailed spectral deconvolution analysis was conducted to identify the probability fractions of the relevant secondary structures including regular and disorder helices and sheets, turns, and unordered folding. The analysis revealed that GH12 and GH12-M2 have similar secondary structure fractions in the absence of TFE, with the regular helix fraction slightly larger (3% versus 0%) for the GH12 peptide. With 10% TFE in the buffer solution, the result is a similar distribution of secondary structure fractions  
Figure 2: (**a**) Mean residue ellipticity (MRE) of far-UV CD Spectra for GH12 and GH12-M2 with varying levels of 2,2,2-trifluoroethanol (TFE). (**b**) Circular dichroism spectra deconvolution through CD Pro analysis. The percentages indicate the amount of TFE in the solution.  
to the peptides in buffer only. For GH12, the addition of 10% TFE to the buffer reduced the potential of the regular helix so that all the categories are within 1-2%. At solutions with 20% TFE, we observed a large difference between the secondary structure fractions for GH12 and GH12-M2, consistent with the original CD spectra results. GH12-M2 resulted in more regular and distorted helical features (38%) compared to GH12 (4%). Regular sheet and distorted sheet fractions were greater for GH12 (44%) versus GH12-M2 (15%). At high TFE concentrations (>20%), the two peptides stabilize in the fractions of their CD deconvolution. The GH12 peptide in solutions containing 40% and greater TFE exhibit a larger turn fraction (21-23%) and smaller helix fraction (37-38%) than calculated for GH12-M2 turns (16-17%) and helix fraction (42-43%).  
In agreement with previous studies, GH12 exhibits ahelix conformations in solvents mimicking interactions at the membranes [76]. Our results are in agreement with the trends that are shared by the modified GH12 and GH12-M2. Due to significant differences in the structural analyses of CD, we did further characterization using Fourier transform infrared (FT-IR) and Raman spectroscopy.