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

Cyclin-dependent kinase 6 (CDK6) is an important protein kinase that regulates cell growth, development, cell metabolism, inflammation, and apoptosis. Its overexpression is associated with reprogramming glucose metabolism through alternative pathways and apoptosis, which ultimately plays a significant role in cancer development. In the present study, we have investigated the structural and conformational changes in CDK6 at varying pH employing a multi-spectroscopic approach. Circular dichroism (CD) spectroscopy revealed at extremely acidic conditions (pH 2.0-4.0), the secondary structure of CDK6 got significantly disrupted, leading to aggregates formation. These aggregates were further characterized by employing Thioflavin T (ThT) fluorescence. No significant secondary structural changes were observed over the alkaline pH range (pH 7.0-11.0). Further, fluorescence and UV spectroscopy revealed that the tertiary structure of CDK6 was disrupted under extremely acidic conditions, with slight alteration occurring in mild acidic conditions. The tertiary structure remains intact over the entire alkaline range. Additionally, enzyme assay provided an insight into the functional aspect of CDK at varying pH; CDK6 activity was optimal in the pH range of 7.0-8.0. This study will provide a platform that provides newer insights into the pH-dependent dynamics and functional behavior of CDK6 in different CDK6 directed diseased conditions, viz. different types of cancers where changes in pH contribute to cancer development.

Introduction

Cyclin-Dependent Kinase 6 (CDK6) is an important regulatory protein of the cell cycle and metabolism [1]. It controls the G1-S phase transition of the cell cycle through the Rb-E2F pathway [2-5]. CDK6 is a key regulator in various cellular processes, such as cell proliferation [6-8], differentiation [9-11], inflammation [12], apoptosis and cancer [1, 8, 13, 14], suggesting the importance of CDK6 [15]. Its activity is directly activated by cyclin activating kinase (CAK) and cyclin D3, whereas indirectly several growth factors like tumor necrosis factor-α (TNF-α), vascular epithelial growth factor, epidermal growth factor(EGF), nerve growth factor, transforming growth factor-β (TGF-β) and cytokines play a role in its activation [15]. It is reported that CDK6 protein expression increases several folds than normal cell growth during tumor and tumor-associated diseases like cancer [16, 17]. In all cancers like colon, breast, lung, prostate, and stomach, the CDK6 expression level is very high, which signals cancerous cells to show resistance against the several drug molecules, chemotherapy and radiotherapy through continuous mutation [18].

In the last few decades, cancer cells show reprogramming of cellular and metabolic pathways. Several reports prove that the increased CDK6 level controls cell proliferation and alters the metabolic pathway of glucose consumption. CDK6 inhibits the Phosphofructokinase (PFK) and Protein Kinase (PK), key regulator enzymes of the glycolysis pathway. On the contrary, it activates the pentose phosphate pathway (PPP) and serine synthesis pathway that cause NADPH production and prevent ROS generation, which ultimately inhibits apoptosis [1, 19–23]. Evidence suggests that the alteration in the expression level of CDK6 involves cancer and is also found in neurodegenerative disorders like Parkinson’s [16, 24–26]. All these reports highlight the importance of CDK6 as a potential therapeutic target to cure several pathological conditions [26], ranging from cancer to neurodegenerative disorders [27, 28].

There are additional domains in CDKs apart from consensus kinase domains. For instance, CDK16, CDK17 and CDK18 comprise a conserved catalytic domain flanked by amino and carboxy-terminal extensions implicated in the binding of cyclins. CDK12 and CDK13 comprise a kinase domain in the center with additional Arg/Ser rich motifs in the amino terminus that serve as docking sites for splicing factors assemblage and as splicing regulators.

CDKs belong to the CMGC group of kinases [29]. Cyclin-dependent kinases (CDKs) are serine/threonine protein kinase that controls the cell cycle [30]. CDKs and their cyclin strictly control every stage of the cell cycle by signaling checkpoints [31]. CDK6 gene is located on human chromosome 7q21.2, which encodes the 326 amino acids ~ 37 kDa cytosolic protein [32]. The human CDK6 structure is a single polypeptide chain that consists of N- terminal (amino-terminal) domain (5–100 amino acids) and C-terminal (Carboxyl terminal) domain (101–309). The N-terminal domain comprises five β strands with one PLSTIRE α- helix. C- terminal domain comprises mainly of α- helix with a small β sheet [33]. The all-inclusive structure of CDK6 is similar to CDK2 [34]. The catalytic site is present between both terminals. Cyclin-D interacts with amino-terminal and changes its structure, partially activating CDK6; CAK through phosphorylation/ dephosphorylation process at the specific site fully activated it [35, 36]. Several factors, including temperature, the strength of the buffer, pH, ions, macro and micro molecules present vicinity of the cell, is responsible for the function, proper folding and 3D structure of the protein [37-39]. Change in the ions concentration disturbs the pH homeostasis, leading to alteration of net charge on protein, ultimately affecting the protein function implicated in the diseased conditions [38]. It is apparent that any change in cellular pH, alters the cellular processes like cell growth, proliferation, and metabolic rate of cells; all these are hallmarks of cancer [40-42].

CDK6 structure and its function are already well reported, but structural and functional characterization at different physiological pH conditions is unknown. Every protein shows its optimum activity at a particular pH. Thus, this study intends to delineate the effect of various pH conditions on the structure and function of CDK6 protein. We have successfully cloned, expressed and purified the CDK6 protein from the bacterial system. The effect of pH on the secondary and tertiary structure of CDK6 was investigated employing multi-spectroscopic methods, fluorescence, UV visible, CD spectroscopies. ThT fluorescence was done to have an insight to characterize the aggregate formation of CDK6. The structural studies were further complemented with the measurement of the kinase activity at various pH.

Materials and methods

Materials

DifcoTM LB broth Miller (Becton Dickinson, Fisher Scientific, Lenexa, KS, USA) was used for bacterial culture. Antibiotic Kanamycin (Sigma, Saint Louis, MO, USA) 50 μg/ ml was used. Isopropyl-β- thiogalactopyranoside (IPTG) was purchased from Calbiochem (USA). Tris-HCl, NaCl, NaOH, glycine, sodium acetate, sodium phosphate monobasic, dibasic, TritonX-100, dichloro-diphenyl-trichloroethane (DDT), lysozyme was procured from Sigma- Aldrich (St. Louis, USA). Ni- NTA column and beads purchased from BioRad (USA).

Cloning, expression and purification

CDK6 protein was successfully cloned, expressed and purified using our well-established protocol as per earlier published literature [43].

Sample preparation

CDK6 protein structure analysis was carried out using a wide range of pH 2.0 to 11.0 buffers. Glycine-HCl buffer was used for pH range 2.0–3.0; sodium acetate buffer was used for pH 4.0–6.0. Tris- HCl buffer was used for pH 7.0–8.0; sodium bicarbonate buffer was used for pH 9.0–10.0; Glycine-NaOH buffer was used for the pH range 11.0. The protein was incubated with a 50mM concentration of different buffers for 5 h at 25°C, and equilibrium was successfully attained before the spectroscopic measurements. All the measurements were carried out in triplicates form.

CD measurements

Circular dichroism (CD) experiment was carried out at Jasco spectropolarimeter (J-1500, Japan) (PTC-517), equipped with a Peltier-type temperature controller to maintain the temperature. Far-UV CD spectra of CDK6 samples at different pH (2.0–11.0) were recorded in a 1 mm path-length cuvette with protein concentration kept at 8 μM. Spectra were recorded in the range of 205–250 nm with a 100 nm/min scan speed and a response time of 1 s [44]. Each spectrum was an average of 3 consecutive scans. The results were expressed as Mean Residue Ellipticity (MRE) in deg cm2 dmol−1 which is defined as: where θobserved is the observed ellipticity in milli degrees, MRW is the mean residue weight of the protein, l being the path length in cm and c being the protein concentration in mg/ml.

Fluorescence measurement

Fluorescence spectra of CDK6 were measured on Jasco spectrofluorometer (FP 6200, Japan) at 25°C ±1; the connected Peltier device-maintained temperature. The protein concentration was kept at 4 μM, and the sample was excited at 280 nm with the emission spectra recorded in the range of 300–400 nm [45]. Both the slit widths were fixed at 5nm and the response was set to medium intensity. All the measurements were carried out in triplicates and reported spectra were taken after subtracting appropriate blanks. For Thioflavin T (ThT) fluorescence, the sample was excited at 440 nm, and emission was recorded at 450–600 nm.

UV/Vis absorbance measurements

UV/visible absorbance spectra of CDK6 were measured on Jasco UV/Vis spectrophotometer (V-660) connected with the Peltier device to maintain the temperature. The incubated sample spectra with a different range of pH buffer (2.0–11.0) were obtained at 240–340 nm by using a 1 cm path length cuvette. A Triplet set of samples were used for the experiment.

Kinase activity assay

To study the effect of pH on the enzymatic activity of CDK6 protein, kinase assay was carried out in the presence of a different range of pH buffers. CDK6 protein (1 μM) sample was prepared in a different range of pH buffers and freshly prepared ATP was added (50 μM) in a reaction volume of 100 μl and incubated for 1hr at 25°C for enzyme activity measurements. Similar reactions were carried out in triplet form for different ranges of pH buffers. To stop the reaction, a malachite green reagent (200 μl) was added into the reaction mixture and incubated for 30 minutes at 25°C for the development of green color. The absorbance of the final product was measured spectrophotometrically at 620 nm.

Results and discussion

The pH of the solution is a vital player in deciding the charge over the protein’s surface, thereby influencing the chemical stability of the protein. Thus, it is apparent that the pH of the solution plays a key role and influences the structural and functional aspects of the proteins [46, 47]. Thus, considering the importance of pH in governing the protein’s functionality, this study intends to delineate the effect of pH on the structure and activity of an important kinase, CDK6.

Effect of pH on the secondary structure

To see the effect of pH on the secondary structure of CDK6, CD spectroscopy was deployed. CD spectroscopy is a sensitive technique routinely used to study changes in protein conformation under different pH conditions [48]. Native CDK6 showed spectral characteristics of a mixture of α-helix and β-sheet structures. The protein retained its native secondary structure in the pH range of 5.0–8.0, as revealed by the far-UV CD spectrum (. However, visible aggregates were observed in the pH range of 2.0–4.0 that interfered in CD spectra and hence almost no dichroic signal was obtained in this pH range. At pH 7.0–8.0, the protein attains a properly folded conformation evident from the far UV CD spectra obtained in this pH range.

Changes in the secondary structure of CDK6 measured by far-UV CD.

(A) Far-UV CD spectra of CDK6 in the pH range of 2.0–8.0 (B) Far UV CD spectra of CDK6 in alkaline pH range (7.0–11.0) (C) A plot of MRE at 222 nm (θ222) as a function of pH.

shows far UV CD spectra of CDK6 in the alkaline range (pH 7.0–11.0). Far UV CD spectra are nearly identical in this range with no significant change suggesting that CDK 6 maintains a native-like conformation across the alkaline range with minimal alterations in the secondary structure over the entire range. shows the plot of [θ]222 versus pH and depicts that no significant perturbation of secondary structure occurs at alkaline pH (7.0–11.0). These observations suggest that CDK6 attains maximum structure, indicating a native-like structure at pH 7.0–8.0.

Effect of pH on the tertiary structure

Intrinsic fluorescence studies were carried out to see the effect of pH on the tertiary structure of irisin. Since intrinsic fluorescence of proteins depends on aromatic amino acid residues [49, 50], a change in intrinsic fluorescence is a sign of change in the local environment of aromatic amino acid residues. Thus, any structural perturbation that affects the microenvironment around the fluorophore is often reflected in terms of changes in the emission spectrum of the protein [51]. CDK6 possesses 8 Tyr and 3 Trp residues, and hence, we have investigated the effect of pH on the tertiary structure of CDK6 in terms of intrinsic fluorescence spectroscopy. A characteristic redshift in the emission maxima is observed when a protein unfolds due to the increased solvent exposure of aromatic amino acid residues. Fluorescence emission spectra of CDK6 in the pH range (pH 2.0–8.0) are depicted in . CDK6 attains its native conformation in the pH range of 7.0–8.0, showing an emission maxima peak at 341 nm, as reported for other kinases [52]. A recently published study also reported that pyruvate dehydrogenase kinase 3 (PDK3) and Sphingosine kinase 1 (SPHK1) maintains their tertiary structure over the alkaline pH range [52, 53]. Another recently published literature reported that irisin, a myokine, maintains its structure in the alkaline pH range [39].

Presenting pH-induced changes in the tertiary structure of CDK6 probed by fluorescence spectroscopy at 25°C.

(A) Fluorescence emission spectra of CDK6 in the pH range of 2.0 to 8.0 (B) Fluorescence emission spectra of CDK6 in the pH range of 7.0–11.0. (C) Changes in emission λmax as a function of pH.

In the pH range of 5.0–6.0, there was no spectral shift in the emission maxima, suggestive of the fact that the environment around the aromatic amino acid residues was not perturbed to a significant extent. However, a decrease in fluorescence intensity was observed attributable to the protonation of water molecules or acidic amino acids surrounding Trp residues that act as dynamic quenchers of intrinsic fluorescence [54]. A spectral shift was obtained in the pH range of 2.0–4.0along with a decrease in the fluorescence intensity suggestive of the tertiary structural perturbations in this pH range. At pH 4.0, a decrease in the fluorescence intensity was coupled with a redshift of nearly 5nm, while at pH 2.0 and 3.0, visible aggregates were seen, and a blue shift of 3 nm and 6 nm was recorded, respectively. CDK6 maintains its native conformation at pH 5.0–8.0.

depicts the fluorescence emission spectra of CDK6 in the alkaline pH range (pH 7.0–11.0). It is quite clear from the figure that CDK6 maintains its tertiary structure in the alkaline range (pH 7.0 to 11.0), attributable to no significant change in λmax across the alkaline range. However, there was an evident decrease in the fluorescence intensity beyond pH 9.0. This decrease is attributable to the deprotonation of basic amino acids present in the vicinity of intrinsic fluorophores causing fluorescence quenching. Protonation/deprotonation of amino acid side chains may lead to charge destabilization in the local environment by disrupting the electrostatic interactions and internal salt bridges present in the native state [55]. The plot of λmax as a function of pH is depicted in ; fluctuations were evident in λmax in the extreme acidic conditions, i.e., in the pH range of 2.0–4.0. In the pH range of 5.0–8.0, no significant change in the emission wavelength maxima was observed, suggesting no changes in the tertiary structure. However, a slight redshift of 2 nm was observed beyond pH 8.0. All these observations reveal that the tertiary structure of CDK6 is maintained in the alkaline pH range. In contrast, significant tertiary structural alterations occurred in the extreme acidic, with slight alterations occurring in the mild acidic conditions.

To further examine the effect of pH on the tertiary structure of CDK6, UV-vis spectroscopy was employed (240–340 nm). Conjugated double bond system present in the side chains of aromatic amino acids acts as a chromophore and absorbs light strongly in the UV region (240–340 nm) [56]. Additionally, the microenvironment of the aromatic residues corresponds to changes in UV absorption maxima; increased solvent exposure causes a blue shift in λmax. shows the UV absorption spectra of CDK6 in the pH range (2.0–8.0). In the pH range of 5.0–8.0, nearly identical UV spectra were obtained, suggesting that minimal tertiary structural alterations occur and protein maintains its native-like conformation. In the pH range of 2.0–4.0, completely distorted UV spectra with a very high scattering were recorded. These distorted spectra suggest the distortion of the tertiary structure of CDK6 in the acidic conditions with subsequent aggregation. On the other hand, no significant changes were observed in the UV spectral profile of CDK6 across the alkaline range (pH 7.0–11.0), implying that CDK6 maintains its native-like conformation in the alkaline pH range.

Showing pH-induced changes in the tertiary structure of CDK6 were investigated by absorbance spectroscopy at 25°C.

(A) Absorption spectra of CDK6 in the pH range of 2.0 to 8.0. (B) Absorption spectra of CDK6 in the pH range of 7.0 to 11.0.

These observations corroborate fluorescence and CD spectroscopy observations advocating that CDK6 maintains its native-like conformation in the alkaline pH range. Under extreme acidic conditions, distortion in its tertiary structure occurs with subsequent aggregation.

Thioflavin T fluorescence

All the above observations suggest that aggregation of CDK6 occurs under extreme acidic conditions. Thus, ThT fluorescence was carried out to strengthen these observations further since ThT is an extrinsic dye routinely employed to characterize aggregates [57]. ThT binds to β-sheets, and in the case of aggregates, ThT-binding to aggregates causes an increase in the fluorescence [58, 59]. shows the ThT fluorescence intensity of CDK6 in the pH range of 2.0–11.0. It is apparent that aggregation occurs at pH 2.0–4.0, while no aggregation is observed at other pH levels. These observations are concurrent with other assays validating that CDK6 aggregation occurs in extremely acidic conditions.

Aggregates characterization by ThT fluorescence.

ThT fluorescence intensity was plotted as a function of pH (2.0–11.0).

Effect of pH on kinase activity

The enzyme assay was carried out to delineate the effect of pH on the functional aspects of CDK6 as per previously published protocols [52, 60]. This assay involves the formation of a green-colored complex that is measured spectrophotometrically at 620 nm. CDK6 shows maximum enzymatic activity in the pH range of 7.0–8.0, suggestive of the fact that it exists in its native conformation. A significant decline in the enzymatic activity was observed moving away from this pH range in both directions (acidic and alkaline). gives an overview of the enzymatic activity of CDK6 at different pH. The maximum activity of CDK6 at pH 8.0 was arbitrarily set as 100% to normalize the data. Our earlier spectroscopic observations revealed that CDK6 maintains its native-like conformation over the alkaline pH range. Thus, this loss in enzymatic activity in this alkaline pH range can be attributed to the ionization states of active site residues, which further affect catalytic activity. In the pH range of 2.0–4.0, CDK6 forms aggregates that interfere in this assay and thus, the enzymatic assay was not recorded at these pH conditions.

The kinase activity of CDK6 was plotted as a function of pH (2.0–11.0).

The maximum activity of CDK6 was observed at pH 8.0, and it was arbitrarily set as 100% to normalize the data. Due to protein aggregation, the enzymatic activity was not determined in the pH range of 2.0–4.0.

Conclusions

It was discovered in this study that pH affects the structure and functional activity of CDK6. It was found that the pH of the solution influenced secondary and tertiary structural alterations in CDK6. 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. Consistent with this finding, maximum ThT fluorescence was observed in the extremely acidic conditions confirming the formation of CDK 6 aggregates under this condition. CDK6 maintains native-like conformation with no secondary structural alterations in the pH range of 5.0–8.0. CDK6 maintains its secondary structure over the entire alkaline range, evident from similar CD spectra.

Fluorescence and UV spectroscopic analysis revealed that CDK6 has a completely disrupted tertiary structure in the extremely acidic conditions. While slight alterations in the tertiary structure of CDK6 are observed in mild acidic conditions. CDK6 maintains its tertiary structure in the alkaline pH range. Enzyme assay revealed that CDK6 showed maximum kinase activity near physiological pH (pH 7.0–8.0), having an optimum pH value of 8.0. The change in the structure of CDK6 based on the pH can be further useful to understand the disease condition and cellular homeostasis to protein function under a variable range of pH conditions.

30 Dec 2021

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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.

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8 Jan 2022

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.

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Response: Thank You. We don’t opt for that.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

25 Jan 2022

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

PONE-D-21-38401R1

Dear Dr. Hassan,

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Reviewer #3: Yes

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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.

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Reviewer #1: No

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3 Feb 2022

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.

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