Designing Collagen-Binding Peptide with Enhanced Properties Using Hydropathic Free Energy Predictions

Round 1

Reviewer 1 Report

The authors reported the use of hydropathic free energy-based screening method to guide the design of a collagen binding peptide (TKKLTLRT) with an enhance stability against different pH environment, and validated the prediction using a quantum-dot based binding assay. While the manuscript is well written, there are few major issues that require the authors’ attention:-  

1. In lines 322-325, the authors stated that the screening method calculated the average hydropathy value to identify the likely binding sites for collagen based on three consecutive amino acid segments, in parallel to codons within nucleic acid sequences. However, the rationale for selecting three consecutive amino acid segments for calculation is unclear. Furthermore, a codon only codes for an amino acid.

2. In line 218, the composition of mobile phase A does not add up to 100%.

3. In Figure 3 legend, it was stated that “The 3-amino acid binding sites are labelled with the peptide sequence when the potential free energy is below -0.693 kcal/mol”. Firstly, there are no such labels in the figure, and secondly, it is unclear why an arbitrary cut-off value of -0.693 kcal/mol was chosen.

4. Both Figure 4 and Figure 5 showed the structure of the LRR-10 and TKKLTLRT peptide, respectively, with the potential binding sites circled. Are these experimentally resolved (X-ray, NMR) structures? If not, in my opinion, these figures (especially with all the rotations) are not really necessary as they do not add any useful input to the text.

5. Both Figure 6 and Figure 7 showed the potential 3-amino acid binding sites between the peptides and collagen. As the plots are showing potential binding sites across collagen residues 1-1300s, are these plots suggesting that the peptides can bind to any sites on collagen? If this is so, what would the plot of the same peptides with other protein (not collagen) looks like? Also, what would the plot of other non-collagen binding peptide with collagen looks like? The authors need to show these additional plots to demonstrate the usefulness of this approach.

6. The authors also did not explain in detail how the location of the additional leucine was decided in TKKLTLRT peptide. This need to be addressed by the authors.

7. Figure 7 also seems to suggest that TKKLTLRT has higher stability in binding to collagen than LRR-10. This, however, was not validated by the quantum-dots binding assay. An additional assay on LRR-10 would certainly strengthen the readers’ confidence on the validity and accuracy of this approach.

Author Response

We included our responses for reviewer 1 in the attached file. 

Author Response File: Author Response.docx

Reviewer 2 Report

Major comments:

The research topic is great and interesting, but the manuscript needs to address some major comments that I have below:

1.       In Fig. 6 legend, the authors have mentioned that “The potential binding sites are yellow for COL1A2 DNA Sense Peptide 462 (TKKTLRT) and pink for collagenase subsequence (SQNPVQP).” I would recommend changing one of the colors or making a shade darker for pink. It would be difficult for the readers to follow the manuscript if the colors are overlapping.

 

2.       I would recommend authors include a sentence about what is -kT in the Y-axis of figs 6 and 7.

Author Response

The attached file includes our responses for Reviewer 2. 

Author Response File: Author Response.docx

Reviewer 3 Report

The Authors have investigated an interesting topic, and the theme has been properly described.

I would like to congratulate the authors for the good quality of the article, the literature reported used to write the paper, and the clear and appropriate structure.

The manuscript is well written, presented and discussed, and understandable to a specialist readership.

In general, the article's organization and structure are satisfactory and in agreement with the journal instructions for authors. The subject is also adequate with the journal's scope.

The work shows a conscientious study in which a very exhaustive discussion of the literature available has been carried out.

The introduction provides sufficient background, and the other sections include results clearly presented and analyzed exhaustively.

As a specific comment,

1.      I suggest trying to present some important results in the abstract section.

2.      I suggest that authors minimize the introduction section, remove the old references, and check if all the references have been cited in the text or reported in the references list.

3.      The finishing of the paper needs some revision as some titles came in italics and some are not.

4.      The results contain more references, and the discussion needs more discussion in the view of the results.

5.      Follow the instruction for the author regarding the references style, “Journal names.”

So, I recommend the acceptance of the paper after minor revision.

Author Response

The attached file includes our responses for Reviewer 3. 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

In their revisions to the manuscript and in their response letter, the authors have attempted to address most of the comments and requests of the reviewers. While the efforts by the authors to provide the additional control experiments are commendable, the results and discussion of the additional work, especially the analysis of the collagen-binding peptides against decorin, did not add confidence to the reliability of the hydropathic free energy method as a quick evaluation method of peptides for binding to other proteins. To illustrate, Figure S4 showed that all the collagen-binding peptides can also bind to decorin. Unless decorin is a known binding target of collagen-binding peptides experimentally, this results only suggest that the method is not able to distinguish a true binder from non-specific binders, and thus the reported observation (on the successful use of this method in designing a collagen binding peptide with enhanced properties) could be merely just by chance. This is not surprising as the authors themselves are also aware that a protein-protein interaction is highly dependent on the folding of the interacting proteins, and not just their sequences alone. 

Other comments include:-

1. Missing Section 3.1 heading

2. Missing plots for pH 2 and pH 9 in Figure 3

 

3. Missing definition for cyan and magenta circles in the Figure legend 7.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

In the revised manuscript and the response to reviewer, the authors have attempted to address the comments from the reviewer by providing additional experimental data. However, the limitation of the method used has raised a serious doubt in the validity of the method in helping the authors to design a more stable peptide binding to collagen, and its usefulness as an enrichment tool to e.g. molecular docking in virtual screening process, as a whole. For these reasons, it is regrettable that this manuscript cannot be accepted at this point.