## 5 Conclusions  
In this study, we developed a screening tool to highlight hydropathy free energy features of three different collagen-binding peptides to rationally engineer a high-stability collagen-binding peptide. We used sequence features from two collagen-binding peptides to rationally engineer a collagen-binding peptide with enhanced binding property. We extended the design of sense/antisense peptides for collagen-binding applications. We provided a hydropathy-based screening method which is pH dependent for peptide-collagen interactions, which is designed to reduce the computational time of virtual screening by reducing the number of molecular docking attempts for false binding cases. Furthermore, we used this method to show the design of a collagen-binding peptide with predicted high stability of binding across a wide range of pH environments. This hydropathy-based strategy is useful for screening peptide designs with less computational effort than molecular docking all interacting candidate subsequences, and it has a low false negative rate. Our scanning method assumes all collagen and peptide residues are available at the surface. This assumption helps to avoid false-negative binding results and allows for the inclusion of surface availability filtering as a separate tool. If the screening calculation time reduces the molecular docking computation time more than the screening calculation time, computational screening will be faster and more accurate. If the screening computational time is less than the reduction of molecular docking computation for true negatives the screening method identifies, it saves computational time to be invested in other virtual screening methods. Therefore, this strategy can serve as an enrichment tool for virtual screening processes where molecular docking can lower the false-positive rate of the design candidates. Virtual screening processes such as AutoDock [62] and structure-based virtual screening (SBVS) approaches [63] would benefit from more focused molecular docking so that more design features can be considered for drug candidates with the same computational resources.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13053342/s1](https://www.mdpi.com/article/10.3390/app13053342/s1)]([https://www.mdpi.com/article/10.3390/app13053342/s1](https://www.mdpi.com/article/10.3390/app13053342/s1)), Figure S1: Evaluation of hydropathic free energy estimates for collagen III-derived peptide examples; Figure S2: Evaluation of hydropathic free energy estimates for candidate high-stability binding peptides TKKTLRT and LRETLNNN; Figure S3: Evaluation of collagenase peptide and DNA complement peptide on decorin; Figure S4: Evaluation of collagenase peptide, DNA complement peptide, high-stability collagen-binding peptide, and LRR-10 on decorin; Figure S5: Evaluation of binding free energy between LRR-10 and decorin at pH 7 by accessibility of decorin residues.  
Supervision, C.T.; computational investigation and analysis, K.B.; experimental analyses, A.K.C. and E.D.; writing--original draft preparation, K.B; editing, A.K.C.; review and editing, C.T. and P.S. All the authors contributed; project administration, P.S. and C.T.; funding acquisition and resources, P.S. and C.T. All authors have read and agreed to the published version of the manuscript.  
**Funding:** Research reported in this publication was supported by the National Institute of Dental & Crani Research of the National Institutes of Health under Award Number R01 DE025476. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.  
**Institutional Review Board Statement:** Not applicable.  
**Data Availability Statement:** Not applicable.  
**Acknowledgments:** The authors would like to thank [PERSON] for his support during the experimental preparations.  
**Conflicts of Interest:** The authors declare no conflict of interest.