PONE-D-22-04782AlphaFold2 and RoseTTAFold Predict Posttranslational modifications. Chromophore Formation in GFP-like Proteins.PLOS ONE

Dear Dr. Marc Zimmer,

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Reviewers' comments:

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Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

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Reviewer #1: Yes

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Reviewer #1: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors report on a very interesting computational approach to figure out whether green fluorescent protein-like proteins fluoresce, based on their amino acid sequence. They use the AlphaFold2 and RoseTTAFold artificial intelligence software which was trained on 180,000 protein structures in the protein data bank to predict the folded protein with atomic resolution. This data base contains 578 GFP-like proteins of which 10 have no chromophore, i.e. do not fluoresce. (It wasn’t clear to me whether they can still absorb, though, like a dark acceptor in FRET?)

The present manuscript presents the amino acid sequences of 44 GFP-like proteins that are not in the protein data bank to AlphaFold2 and RoseTTAFold, and the software calculates the 3-dimensional structure of these proteins to atomic resolution. While the precise solid-state structure of these 44 proteins has not been determined experimentally, importantly, it is known from experiment whether they fluoresce or not.

The software correctly folds the amino acid sequences into a fluorophore for the fluorescent proteins, and no fluorophore for the ones that do not fluoresce, if I understand this correctly.

I think is a phenomenal result, and the link to experiment via fluorescence is ingenious – it requires no details apart from “yes” or “no” of fluorescence, and no details of spectra or lifetime, which essentially makes it a powerful binary approach (0 or 1, so to speak).

The manuscript is very well written and easy to follow. The work will be of interest to the research community developing (and also using) fluorescent proteins, and those predicting protein structure from amino acid sequences in general. It will also be of interest for researchers in artificial intelligence, machine learning and other computational and computer-simulation approaches.

I only have some minor comments:

1) Maybe a figure with a representative structure of a GFP-like protein with fluorophore investigated in this study would be useful, as well as such a protein without a fluorophore.

2) By chromophore, the authors mean a structure that fluoresces, not one that just absorbs light, is that correct?

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Reviewer #1: No

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AlphaFold2 and RoseTTAFold Predict Posttranslational modifications. Chromophore Formation in GFP-like Proteins.

PONE-D-22-04782R1

Dear Dr. Marc Zimmer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Maria Gasset, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data 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 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—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Now I agree that a figure of a fluorescent and non-fluorescent GFP would be of limited use, as it is dominated by the barrel, not the all-important fluorophore. I leave it to the authors to decide whether to include this figure or not, it may be helpful for non-experts in the field. The chromophore definition is definitely useful, and I look forward to seeing this nice work published.

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7. 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: No