Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

Takashi Yoshida; Norihisa Yasui; Yuko Kusakabe; Chiaki Ito; Miki Akamatsu; Atsuko Yamashita

doi:10.1371/journal.pone.0218909

Peer Review History

Original SubmissionJune 10, 2019
21 Aug 2019 Decision Letter - Piero Andrea Temussi, Editor PONE-D-19-16465 Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3 PLOS ONE Dear Dr. Yamashita, 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. We would appreciate receiving your revised manuscript by Oct 05 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 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 ****** 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 ****** 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: The manuscript "Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3" presents interactions between various amino acids and taste receptor by differential scanning fluorimetry. This work is another important step how amino acids interact with taste receptor. However, the lack of detailed conditions in DSF analysis, the reviewer concluded this manuscript is not recommended to publish in the present form. This manuscript would be strengthened by a more careful and detailed description of the methods as well as results as explained below. As shown in the Figure 1A, two potential binding sites for amino acids seem to exist in the receptor, however, no detailed comments are presented from DSF results. The amino acids ligands truly bind two sites? The authors should be clarified the binding specificity of ligands as to each site including DSF results. In Figure 1B (Gln), and Figure 2A (Ala, Glu, Arg), one minor peak, around 50�C is visible. What is this minor peak? As the peak is more obvious as ligands concentration increases, some other effects such as a pH change might be involved. The authors should be clearly addressed these issues. Several other points that should be addressed: In DSF experiments, the authors use Tris buffer. Generally, this buffer is suspected to induce pH change when temperature increases. The reviewer thinks DSF results also might be influenced by pH change as Tm values also seem to be sensitive to type of amino acids. I could not judge from the present manuscript, though, the pH value with each ligand, especially at high concentration, should be indicated in the manuscript. Figure 2B Label typo for Glu The reviewer also thinks this manuscript would become more attractive if possible negative controls were included (amino acids ligand which does not bind or weakly binds receptor since high concentration amino acids ligands seems to be involved in non-specific binding to receptor. ****** 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 [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0218909.r001
Revision 1
2 Sep 2019 Author Response Responses to Reviewer Reviewer #1: The manuscript "Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3" presents interactions between various amino acids and taste receptor by differential scanning fluorimetry. This work is another important step how amino acids interact with taste receptor. However, the lack of detailed conditions in DSF analysis, the reviewer concluded this manuscript is not recommended to publish in the present form. This manuscript would be strengthened by a more careful and detailed description of the methods as well as results as explained below. Reply: We appreciate the reviewer’s valuable comments on our manuscript. We added descriptions in the sections pointed by the reviewer, as described below. As shown in the Figure 1A, two potential binding sites for amino acids seem to exist in the receptor, however, no detailed comments are presented from DSF results. The amino acids ligands truly bind two sites? The authors should be clarified the binding specificity of ligands as to each site including DSF results. Reply: We added the discussion about the presence of two potential binding sites in the protein and the DSF results in this study at l. 370-374, p. 17. In Figure 1B (Gln), and Figure 2A (Ala, Glu, Arg), one minor peak, around 50�C is visible. What is this minor peak? As the peak is more obvious as ligands concentration increases, some other effects such as a pH change might be involved. The authors should be clearly addressed these issues. Reply: We have a section discussing about the biphasic melting transition of the protein, indicated by the presence of two peaks (two maxima) in their derivatives, at “Thermodynamic properties of T1r2a/T1r3LBD” in Discussion. In the last paragraph of this section, we discussed about the melting transition around 50 ˚C, corresponding to the peak around 50 ˚C in the derivatives pointed by the reviewer. To make this more explicit, we added the notions correlating the melting transition with the maxima in their derivatives at l. 195-199, p. 9-10, and l. 345-346 in p. 16. In DSF experiments, the authors use Tris buffer. Generally, this buffer is suspected to induce pH change when temperature increases. The reviewer thinks DSF results also might be influenced by pH change as Tm values also seem to be sensitive to type of amino acids. I could not judge from the present manuscript, though, the pH value with each ligand, especially at high concentration, should be indicated in the manuscript. Reply: We confirmed that the use of HEPES buffer, one of the buffers with more stable pH upon temperature increase, gave consistent Tm shifts with those observed in this study for several representative amino acid ligands. We added this information at l.116-118, p. 6 and S1 Fig in the revised manuscript. S1 Fig and S2 Fig in the original manuscript were renamed as S2 Fig and S3 Fig accordingly. For the experiments in this study, we adjusted the pH of the amino acid solution to ~ 8.0 before mixing with the protein, thus the pH change by the addition of the amino acid was unlikely. We added the detailed description of the preparation of the assay solution at l. 106-109, p. 6. Figure 2B Label typo for Glu Reply: We corrected the label. The reviewer also thinks this manuscript would become more attractive if possible negative controls were included (amino acids ligand which does not bind or weakly binds receptor since high concentration amino acids ligands seems to be involved in non-specific binding to receptor. Reply: We already have a negative control, D-alanine, exhibiting no substantial Tm shifts by addition of up to 10 mM, the highest amino-acid concentration tested in this study, as shown in Fig. 3A and described at l.257-261, p. 12. Attachments Attachment Submitted filename: Response2reviewers_190829.docx https://doi.org/10.1371/journal.pone.0218909.r002
18 Sep 2019 Decision Letter - Piero Andrea Temussi, Editor Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3 PONE-D-19-16465R1 Dear Dr. Yamashita, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Piero Andrea Temussi Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0218909.r003
Formally Accepted
23 Sep 2019 Acceptance Letter - Piero Andrea Temussi, Editor PONE-D-19-16465R1 Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3 Dear Dr. Yamashita: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Piero Andrea Temussi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0218909.r004

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