Effect of pH on the structure and function of cyclin-dependent kinase 6

Mohd Yousuf; Anas Shamsi; Farah Anjum; Alaa Shafie; Asimul Islam; Qazi Mohd Rizwanul Haque; Abdelbaset Mohamed Elasbali; Dharmendra Kumar Yadav; Md. Imtaiyaz Hassan

doi:10.1371/journal.pone.0263693

Peer Review History

Original SubmissionDecember 4, 2021
30 Dec 2021 Decision Letter - Shailza Singh, Editor PONE-D-21-38401Effect of pH on the structure and function of cyclin-dependent kinase 6PLOS ONE Dear Dr. Hassan, 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 Feb 11 2022 11:59PM. 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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Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: No Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: Yes ****** 5. Review Comments to the Author Reviewer #1: This study aims to understand the effect of various pH conditions on the structure and function of CDK6 protein. The authors have successfully cloned, expressed, and purified the CDK6 protein from the bacterial system in this study. Furthermore, the effect of pH on the secondary and tertiary structure of CDK6 was investigated employing a multi-spectroscopic approach viz. Fluorescence, UV visible, CD spectroscopy. Overall, the manuscript was very well written. Moreover, the authors used various techniques and interpreted the results appropriately; therefore, the manuscript can be accepted with the following minor comments. 1. The authors can include some more recent references in the introduction section of the manuscript. 2. The authors can also include the 3D structure CDK in the introductory section and highlights the different functional domains. 3. The authors can also briefly include the future perspectives under the conclusion section. 4. The authors can provide the N and C terminus as Italics. 5. Some editing for the English language is required throughout the manuscript due to a few grammatical mistakes. 6. The authors can also include the CD in the abbreviation section. Reviewer #2: The authors have studied structure function relationship of CDK6 at different pH conditions. I have some concerns and comments on the Figures mainly: 1. Figure 1 pH 4 shows no 0 absorbance in the CD spectra, the authors need to explain a bit more as to why this anomaly was observed? The experiments at each pH should be preferably done in triplicates and error bars for the data should be shown. 2. The Figure 1 is it raw data or was the CD spectra of the buffer subtracted to normalize it? 3. Since there is a drastic change between pH 4 and 5, the authors should definitely do two experiments at 4.4 and 4.8 pH to see how the curve changes, else there is no physical significance of this abrupt jump. 4. Figure 2A legends are missing only 3 pH value reported in fig what about the other pH values? 5. fluorescence intensity should be 300X10to the power something, it cannot be just 100.200 etc. Please remove negative fluorescence intensity from the plot. It makes no sense to show negative fluorescence data. 6. Was the fluorescence intensity normalized? If not then authors need to normalize their data. 7. Figure 3 Absorbance > 1 is a DIRECT VIOLATION of BEER LAMBERT's law. The authors should read on this and replot the UV data. Also the experiments should be done in triplicates for robust statistical analyses. A general comment on Page 11 authors discuss perturbation in tertiary structure of CDK6. They specifically use the term "distortion". This is a generalized term. How did they measure distortion? What was the parameter that measured degree of distortion in the 3D structure is not clear at all. Also they may use SEM images to visualize aggregates formed. It is tough to base conclusions without adequate support. I feel with these comments answered, the paper would be much more refined. Reviewer #3: The paper "Effect of pH on the structure and function of cyclin-dependent kinase 6 " work's concept and approach are presented in a convincing manner. The study is adequate, and the PH experiments used are standard and well-established procedures. There are, however, a few comments and questions that must be addressed before the manuscript is accepted for publication. 1. It is necessary to compare this to other kinases in order to determine whether the tertiary structure is preserved throughout the entire alkaline range. 2. A significant structural perturbation occurs in extremely acidic conditions, whereas only minor structural alterations occur in mild acidic conditions, according to the findings. The particle size distribution of protein in acidic conditions can be validated by the author using Dynamic light scattering (DLS) technology. 3. The classification and numbering of headings and subheadings should be verified. 4. Thorough language editing is necessary to improve the presentation's quality. Numerous spacing errors must be corrected throughout the manuscript. In some instances, the use of prepositions is incorrect; please correct. 5. Abbreviations should be double-checked throughout the entire manuscript. They must first be defined before they can be used. As soon as the description is complete, the rest of the manuscript should be written entirely in abbreviations. Many sentences are unclear in places, such as the following: This study delineated the effect of pH on the structure and functional activity...................................subsequently forming aggregates in this pH range. It should be write It was discovered in this study that pH has an effect on the structure and functional activity of CDK6. It was discovered that the secondary and tertiary structural alterations in CDK6 were influenced by the pH of the solution. CDK6 has a disrupted secondary structure when exposed to extreme acidic conditions (pH 2.0-4.0), resulting in the formation of aggregates in this pH range, according to our findings. ****** 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 Reviewer #3: 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. 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Revision 1
8 Jan 2022 Author Response Response to Reviewers' comments Journal: PLOS ONE Manuscript ID: PONE-D-21-38401 Title: Effect of pH on the structure and function of cyclin-dependent kinase 6 Reviewer #1 Reviewer #1: This study aims to understand the effect of various pH conditions on the structure and function of CDK6 protein. The authors have successfully cloned, expressed, and purified the CDK6 protein from the bacterial system in this study. Furthermore, the effect of pH on the secondary and tertiary structure of CDK6 was investigated employing a multi-spectroscopic approach viz. Fluorescence, UV visible, CD spectroscopy. Overall, the manuscript was very well written. Moreover, the authors used various techniques and interpreted the results appropriately; therefore, the manuscript can be accepted with the following minor comments. Response: Thanks for appreciating the scientific content and quality of the manuscript. We have addressed all the suggestions in the revised version of the manuscript. 1. The authors can include some more recent references in the introduction section of the manuscript. Response: Thanks!! We have updated the Introduction section citing latest references. 2. The authors can also include the 3D structure CDK in the introductory section and highlights the different functional domains. Response: We have updated the relevant section in the revised manuscript. 3. The authors can also briefly include the future perspectives under the conclusion section. Response: We have updated the relevant section in the revised version of the manuscript. 4. The authors can provide the N and C terminus as Italics. Response: Thank you for the suggestion. We have updated it in the revised version of the manuscript. 5. Some editing for the English language is required throughout the manuscript due to a few grammatical mistakes. Response: We have proofread the entire version of the manuscript using GRAMMARLY to remove all the grammatical errors. 6. The authors can also include the CD in the abbreviation section. Response: We have added the same in the abbreviations section. Reviewer #2: The authors have studied structure function relationship of CDK6 at different pH conditions. I have some concerns and comments on the Figures mainly: Response: Thanks for reading and evaluating my work. We have addressed all the suggestions in the revised manuscript. 1. Figure 1 pH 4 shows no 0 absorbance in the CD spectra, the authors need to explain a bit more as to why this anomaly was observed? The experiments at each pH should be preferably done in triplicates and error bars for the data should be shown. Response: We agree with the reviewer. The experiments were carried out in triplicates and the error bars have been presented. We have updated the figures. No CD signal was shown because visible aggregates were present that interfered in CD spectrosocopy and hence no signal was obtained. To avoid any confusion, we have removed the CD spectra of pH 2.0-4.0 as no CD signal was obtained. 2. The Figure 1 is its raw data or was the CD spectra of the buffer subtracted to normalize it? Response: All the reported spectra here are the subtracted spectra. 3. Since there is a drastic change between pH 4 and 5, the authors should definitely do two experiments at 4.4 and 4.8 pH to see how the curve changes, else there is no physical significance of this abrupt jump. Response: We observed aggregates in the range of pH 2.0-4.0 and hence, in this range we observed shifts coupled with changes in the intensity. Moreover, pH 5 is comparable to pH 7.0 and 8.0 as there is no shift obtained in this pH region. The abrupt behaviour of pH 4.0 is attribute to the presence of visible aggregates. 4. Figure 2A legends are missing only 3 pH value reported in fig what about the other pH values? Response: It was a formatting mistake and we have rectified. We have updated all the Figures. 5. fluorescence intensity should be 300X10to the power something, it cannot be just 100.200 etc. Please remove negative fluorescence intensity from the plot. It makes no sense to show negative fluorescence data. Response: Thank you!! We have modified all the figures. The negative axis was shown as the pH 2.0 is showing fluorescence intensity in negative range due to presence of aggregates. 6. Was the fluorescence intensity normalized? If not then authors need to normalize their data. Response: All the spectra reported here are the subtracted spectra. 7. Figure 3 Absorbance > 1 is a DIRECT VIOLATION of BEER LAMBERT's law. The authors should read on this and replot the UV data. Also, the experiments should be done in triplicates for robust statistical analyses. Response: We agree with the reviewer. This is a general phenomenon observed due to very high scattering associated with aggregates. We are aware of the fact that absorbance > 1 is not considered as per BEER LAMBERT law but this was just depicted to show the scattering occurring due to presence of aggregates and confirming the presence of aggregates. 8. A general comment on Page 11 authors discuss perturbation in tertiary structure of CDK6. They specifically use the term "distortion". This is a generalized term. How did they measure distortion? What was the parameter that measured degree of distortion in the 3D structure is not clear at all. Also they may use SEM images to visualize aggregates formed. It is tough to base conclusions without adequate support. Response: Thank you for the suggestion. The term perturbation was used in respect to changes in secondary and tertiary structure observed from CD and fluorescence spectroscopy, respectively. No distortion was observed; we are taking only about structural alterations. Reviewer #3: The paper "Effect of pH on the structure and function of cyclin-dependent kinase 6 " work's concept and approach are presented in a convincing manner. The study is adequate, and the PH experiments used are standard and well-established procedures. There are, however, a few comments and questions that must be addressed before the manuscript is accepted for publication. Response: Thank you for the appreciation of the work and scientific merit of the study. We have addressed all the concerns in the revised version of the manuscript. 1. It is necessary to compare this to other kinases in order to determine whether the tertiary structure is preserved throughout the entire alkaline range. Response: Thank you for the suggestion. We have compared other kinases and updated the section in the revised manuscript. 2. A significant structural perturbation occurs in extremely acidic conditions, whereas only minor structural alterations occur in mild acidic conditions, according to the findings. The particle size distribution of protein in acidic conditions can be validated by the author using Dynamic light scattering (DLS) technology. Response: We agree with the reviewer that structural perturbation occurs in extremely acidic conditions, whereas only minor structural alterations occur in mild acidic conditions as proved by CD and fluorescence spectroscopy. 3. The classification and numbering of headings and subheadings should be verified. Response: Thank You!! The entire manuscript has been proofread to rectify all the mistakes. 4. Thorough language editing is necessary to improve the presentation's quality. Numerous spacing errors must be corrected throughout the manuscript. In some instances, the use of prepositions is incorrect; please correct. Response: We have used advanced version of GRAMMARLY to remove all the grammatical errors and typological errors. 5. Abbreviations should be double-checked throughout the entire manuscript. They must first be defined before they can be used. As soon as the description is complete, the rest of the manuscript should be written entirely in abbreviations. Many sentences are unclear in places, such as the following: This study delineated the effect of pH on the structure and functional activity...................................subsequently forming aggregates in this pH range. It should be write It was discovered in this study that pH has an effect on the structure and functional activity of CDK6. It was discovered that the secondary and tertiary structural alterations in CDK6 were influenced by the pH of the solution. CDK6 has a disrupted secondary structure when exposed to extreme acidic conditions (pH 2.0-4.0), resulting in the formation of aggregates in this pH range, according to our findings. Response: Thanks for the suggestion. We have proofread the manuscript and made the relevant changes as per reviewer’s suggestion. 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. Response: Thank You. We don’t opt for that. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0263693.r002
25 Jan 2022 Decision Letter - Shailza Singh, Editor Effect of pH on the structure and function of cyclin-dependent kinase 6 PONE-D-21-38401R1 Dear Dr. Hassan, 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, Shailza Singh, 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 Reviewer #2: All comments have been addressed Reviewer #3: All comments have been addressed ******** 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 Reviewer #2: Partly Reviewer #3: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A Reviewer #3: Yes ****** 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 Reviewer #2: Yes Reviewer #3: Yes ****** 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 Reviewer #2: Yes Reviewer #3: 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: Authors addressed all the comments raised by the reviewers, therefore manuscript can be accpeted for publication. Reviewer #2: (No Response) Reviewer #3: All the the comments are addressed in a fair way. So, please let the article be published. All the the comments are addressed in a fair way. So, please let the article be published, and thank you for reading it. ****** 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 Reviewer #2: No Reviewer #3: No https://doi.org/10.1371/journal.pone.0263693.r003
Formally Accepted
3 Feb 2022 Acceptance Letter - Shailza Singh, Editor PONE-D-21-38401R1 Effect of pH on the structure and function of cyclin-dependent kinase 6 Dear Dr. Hassan: 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. Shailza Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263693.r004

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