Dear Dr Wood,

Thank you very much for submitting your manuscript "Computational Design of Periplasmic Binding Protein Biosensors Guided by Molecular Dynamics" for consideration at PLOS Computational Biology.

As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Alexander MacKerell

Academic Editor

PLOS Computational Biology

Nir Ben-Tal

Section Editor

PLOS Computational Biology

***********************

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: O’Shea et al. has demonstrated how investigation of protein dynamics provides useful information that can be used to design fluorescence based biosensors. While the overall results are convincing and supported by experimental results, I believe a few more analysis can greatly improve the quality of the manuscript.

1. On page 8, the author writes "we ran 10 x 200 ns of these apo simulations, observing closed-to-open transition in 8/10 of them,". It is a bit vague what criteria authors have used to make this decision. Please provide a quantitative description to support this statement.

2. In Figure 2, panel E, caption, the authors write that they show a "representative" model for open or closed state. How the model was selected, please provide a quantitative description.

3. In figure 2, authors show RMSD compared to the starting structure. How does the RMSD looks like compared to the open state?

4. The manuscript seems to be missing a "Conclusion section". Please provide one.

Reviewer #2: The paper suggests that residue position can add to the insights from backbone dihedral angles to generate additional PBP-based biosensors. However, the paper’s major data point is suspect, and the presentation needs improvement.

Data:

Experiments: one week’s experiments would probably help. Especially for residue 230—the star of the paper-- the paper’s point is suspect because (a) the counts are so small, and (b) the only increase measured occurs at 100 mM—roughly 100-fold greater than precited from Ref. 10. Because 230 is in the binding pocket for maltose (again, ref (10)), one suspects that mutating this residue violates the authors’ “agnostic” principle—which was never intended to extend to the substrate binding site. I suspect that authors have constructed a defective binding site. It will be necessary to purify the protein—a simple task if the construct includes a His6 sequence. A full dose-response relation should follow.

Explanations:

The paper ought to better explain the calculations that underlie the suggestions for experiments. reader The final intelligible sentences is ‘We defined a “contact” . . . .’ After that, the become mysterious to the non-cognoscenti. The mysterious concepts include “pixel”, “pixel clustering”, and the residue-level metrics being correlated with each other. Are you simply asking which residues move together (positive) and which move separately? It would be good to say so. GITHUB is not peer-reviewed, so we don’t whether its explanations are clear.

It would also be good to state the hardware used to perform he simulations and to analyze the data, so the reader knows whether (in 2024) to perform the analyses using a phone, a tablet, a laptop, a desktop, or a supercomputer.

Additional confusion: the matrix of 3D is symmetrical, but the figure does not state this and shades only along the y-axis. And the correlation color map is puzzlingly congruent with the Y-axis. If the authors give though to the poor reader, this would solve many of the paper’s problems in presentation.Some of the paper’s puzzling features would improve if the experimentalists and computationalists simply talk with each other. Thus, what some of the authors call “raw fluorescence”, others call F0. What one set of authors called “uptick” another set would term, “dose-response relation”.

Pedantic corrections:

“The Pearson Correlation Coefficient matrix can be accessed from only the holo crystal structure,”. Is a very confusing statement, in part because “only” has many interpretations. Perhaps it means, “Knowledge the holo structure is sufficient to begin MD simulations, without the necessity for knowing the apo structure.”

Here is the correct capitalization: Person correlation coefficient. Pearson is a proper noun; his correlation coefficient is not. Although one might capitalize the abbreviation (PCP), one does not capitalize the individual words.

**********

Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No: Please upload the raw simulation trajectories to any open source data sharing services.

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Nandan Haloi

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr Wood,

Thank you very much for submitting your manuscript "Computational Design of Periplasmic Binding Protein Biosensors Guided by Molecular Dynamics" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Alexander MacKerell

Academic Editor

PLOS Computational Biology

Nir Ben-Tal

Section Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have fully addressed the concerned raised.

Reviewer #2: This critique repeats some of my original comments. Please take additional care to respond to these comments. Part of our profession is efficient communication. At present, some aspects require the reader to work unnecessarily hard.

Figure 3D still needs work. At present, it’s a square. It should be simply an equilateral right triangle (the lower right half of the present square). The suggested tactic would eliminate the redundant, confusing part. The suggested tactic would eliminate the necessity to explain (to stupid readers like me) why the shaded vertical bars are not also shaded horizontal bars. Again, the fact that the color lookup table has the same height as the vertical axis is also confusing. Please make the color lookup table smaller than the vertical axis, so that harried readers (like myself) don’t have to ask whether the lookup table encodes the vertical axis (it does not!).

Th Journal’s web site does not allow me to view the supplementary material. Therefore, I cannot tell whether the authors have rectified the “F0” vs “raw fluorescence” terminology.

FWIW, what’s the cost of the computer time, the time awaiting intermediate steps in the computational pipeline, and the like? Compared with the cost of time and materials for one round of site-saturated mutagenesis? Is this comparison not even worth considering?

**********

Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: None

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Nandan Haloi

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr Wood,

We are pleased to inform you that your manuscript 'Computational Design of Periplasmic Binding Protein Biosensors Guided by Molecular Dynamics' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Alexander MacKerell

Academic Editor

PLOS Computational Biology

Nir Ben-Tal

Section Editor

PLOS Computational Biology

***********************************************************