A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes

Melanie Ernst; Tugba N. Ozturk; Janice L. Robertson

doi:10.1371/journal.pone.0280693

Peer Review History

Original SubmissionAugust 3, 2022
14 Sep 2022 Decision Letter - Sabato D'Auria, Editor PONE-D-22-21829A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranesPLOS ONE Dear Dr.Janice Lee Robertson, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by October 6, 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "The Robertson lab is supported by the National Institute of General Medical Science, National Institutes of Health (R01GM120260). " We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "ME, TNO and JLR were supported with a grant (R01GM120260) from the National Institutes of General Medical Sciences, National Institutes of Health. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "This study describes a novel analysis approach that is applied to data previously published in Chadda et al., JGP 2018 and Chadda et al., eLife 2016." Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. 4. Please 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. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. 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 Reviewer #2: Yes ****** 4. 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 Reviewer #2: Yes ****** 5. 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: This interesting study addresses a very important point, which is the quantification of fluorophore labeling, in experiments involving protein oligomerization within biological membranes. This methodology might considerably improves the accuracy of the description (i.e. the determination of the dissociation constant) of protein oligomerization in lipid bilayers. Certainly, the application of this method is not very easy and presents some limits, but I think that the authors have carefully addressed this point in the introduction and in the discussion sections. The high quality of the analysis presented for the case study (homodimeric CLC-ec1) makes this paper certainly suitable for publication in PLOS ONE. Reviewer #2: The manuscript by Melanie Ernst et al. entitled “A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes” reports the development of a method to determine fluorophore labeling yields from single-molecule photobleaching data. The authors, based on them previous work with CLC-ec1 (single-molecule photobleaching subunit capture approach) developed a new method to evaluate the fluorophore labelling yields, the stoichiometry and KD values for a range of other CLC-ec1 membrane proteins. The authors combined a single-molecule photobleaching TIRF microscope measures with a computational method to predict the labeling yields. This work demonstrates an approach to measuring the fluorescent labeling yield directly from an experiment in real-time using a known dimeric control, representing a significant advance in the ability to determine fixed or dynamic stoichiometry for membrane proteins in membranes. The authors declare also some constrains: “the fluorophore labelling yields can be determined accurately with single-molecule amounts of well-established dimer controls greatly simplifying the quantitative requirements to study dynamic protein oligomerization” The aim of this work is interesting (and this reviewer appreciates it), the paper is well done in almost all parts. The developed method and experiments performed are interesting (and this reviewer appreciates it), the protocol is well described in all parts. The manuscript in the present form demands a light revision before it can be published, so this reviewer suggests a minor revision of the paper. The paper should be modified in some parts. Major issues: 1) The abstract is too much long and results not clear, please reduce and improve. 2) Please adds the errors as bar in all graphs reported (i.e., Fig. 1 panel C is missed), 3) Please adds (in materials and methods) a short paragraph that describe the statistical analysis used, 4) Please divide the Discussion section in two sections (Discussion and Conclusion) 5) Which is the operational range of your methods? Please explicit the constrains and limits in the conclusion Minor issue: 1) Please revise the text for some misspelling ****** 6. 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 Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0280693.r001
Revision 1
1 Dec 2022 Author Response We thank the reviewers for their considerate feedback and have revised the proposal accordingly. We believe the updated manuscript is significantly improved. Here is a detailed summary of our responses: Reviewer #1: This interesting study addresses a very important point, which is the quantification of fluorophore labeling, in experiments involving protein oligomerization within biological membranes. This methodology might considerably improves the accuracy of the description (i.e. the determination of the dissociation constant) of protein oligomerization in lipid bilayers. Certainly, the application of this method is not very easy and presents some limits, but I think that the authors have carefully addressed this point in the introduction and in the discussion sections. The high quality of the analysis presented for the case study (homodimeric CLC-ec1) makes this paper certainly suitable for publication in PLOS ONE. Reviewer #2: The manuscript by Melanie Ernst et al. entitled “A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes” reports the development of a method to determine fluorophore labeling yields from single-molecule photobleaching data. The authors, based on them previous work with CLC-ec1 (single-molecule photobleaching subunit capture approach) developed a new method to evaluate the fluorophore labelling yields, the stoichiometry and KD values for a range of other CLC-ec1 membrane proteins. The authors combined a single-molecule photobleaching TIRF microscope measures with a computational method to predict the labeling yields. This work demonstrates an approach to measuring the fluorescent labeling yield directly from an experiment in real-time using a known dimeric control, representing a significant advance in the ability to determine fixed or dynamic stoichiometry for membrane proteins in membranes. The authors declare also some constrains: “the fluorophore labelling yields can be determined accurately with single-molecule amounts of well-established dimer controls greatly simplifying the quantitative requirements to study dynamic protein oligomerization” The aim of this work is interesting (and this reviewer appreciates it), the paper is well done in almost all parts. The developed method and experiments performed are interesting (and this reviewer appreciates it), the protocol is well described in all parts. The manuscript in the present form demands a light revision before it can be published, so this reviewer suggests a minor revision of the paper. The paper should be modified in some parts. Major issues: 1) The abstract is too much long and results not clear, please reduce and improve. Thank you for pointing this out. We have revised the abstract to elaborate on the results to hopefully make this clearer. In addition, the abstract is now shortened to 237 words, adhering to the journal guidelines. 2) Please adds the errors as bar in all graphs reported (i.e., Fig. 1 panel C is missed), We revised the figures to improve visibility of error bars that were not visible in the previous version. Originally, we did not include error bars in Fig 1C because these plots reflect model data derived from a mathematical simulation. However, since this is a stochastic model where we randomly simulate protein partitioning into liposomes, we can report the variability associated with the results, even though the standard deviation in the data are small. We have now modified Fig 1C to include these error bars that reflect the simulation variability. 3) Please add (in materials and methods) a short paragraph that describe the statistical analysis used, In the “Materials and methods” section, we have added a final section titled "Statistical analyses" that describes the type of errors calculated throughout, and the statistical tests that we used (page 12-13, lines 322-459). In addition, we modified Fig 4C to report mean ± sem from independent KD estimations per sample, rather than the standard deviation of the parameter estimation. The new representation allows for statistical testing between means that will be more useful for readers of this study. 4) Please divide the Discussion section in two sections (Discussion and Conclusion) The discussion is now divided into "Discussion" and "Conclusion" sections. 5) Which is the operational range of your methods? Please explicit the constrains and limits in the conclusion This is an excellent question and we thank the reviewer for asking this as we neglected to include this important result in the previous version of our manuscript. We have now revised the paper to include a new Fig 5, which shows a statistical analysis of the operational range for these studies based on the ability to discriminate monomer and dimer photobleaching probability distributions. With this, we conclude that for mole fraction densities of χ = 10-9 to 10-5 subunits/lipid, that Pfluor � 0.7 and Pbg � 0.1 allow for significant discrimination between populations. However, if the reaction can be well described for χ � 10-6 subunits/lipid, then the labeling conditions can be lowered to Pfluor � 0.4, Pbg � 0.1. Despite this, we comment that labeling should be optimized as close to Pfluor to 1 and Pbg to 0 to increase robustness in this photobleaching approach. This analysis is now described in a new section of the results on page 21-22, lines 741-826, Fig 5, and in the “Conclusions” section on page 26, lines 1030-1036. Minor issue: 1) Please revise the text for some misspelling We have now carefully read through the manuscript and revised the text for misspelling. Attachments Attachment Submitted filename: Response-to-Reviewers-Ernst,Ozturk,Robertson-2022.11.03.pdf https://doi.org/10.1371/journal.pone.0280693.r002
6 Jan 2023 Decision Letter - Sabato D'Auria, Editor A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes PONE-D-22-21829R1 Dear Dr. Janice Lee Robertson, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Sabato D'Auria Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0280693.r003
Formally Accepted
12 Jan 2023 Acceptance Letter - Sabato D'Auria, Editor PONE-D-22-21829R1 A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes Dear Dr. Robertson: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sabato D'Auria Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0280693.r004

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