The expression vector pET-28a(+) and E. coli BL21 (DE3) competent cells were obtained from Qiagen, while E. coli DH5α cells came from Invitrogen. Analytical-grade routine reagents including NaCl and EDTA were purchased from Merck (India), LB broth from Merck (Darmstadt, Germany), kanamycin and IPTG from Sigma-Aldrich, and Ni–NTA purification materials from Bio-Rad and Qiagen.

Limonene used in the study was purchased from Sigma-Aldrich (USA).

Docking was performed between limonene (Conformer3D_COMPOUND_CID_22311) and CDK6 crystal structure 3NUP using InstaDock. Binding-affinity calculations were carried out with QuickVina-W, a modified version of AutoDock Vina, and a blind-docking strategy was used to explore potential ligand-binding sites across the protein surface.

The paper explicitly defines the thermodynamic relationships used to derive predicted inhibitory constant (Ki,pred), pKi, and ligand efficiency (LE) from the docking-derived binding free energy.

Overnight bacterial culture was pelleted, resuspended, lysed, and treated with proteinase K. Plasmid purification was performed using phenol-chloroform extraction, and DNA concentration and purity were checked by Nanodrop at 260 nm.

The isolated plasmid was also confirmed by agarose gel electrophoresis.

A 0.8% agarose gel was prepared in TAE buffer, ethidium bromide was added after cooling, and samples mixed with bromophenol blue loading dye were loaded into wells. The gel was run at 100–150 V for approximately 45–60 minutes and visualized on a UV transilluminator using a gel documentation system.

A single colony was grown in LB with kanamycin, expanded into fresh medium, and cultured until OD600 reached 0.4–0.6. Cells were chilled on ice, centrifuged, resuspended in ice-cold 10% glycerol, aliquoted, and stored at −80 °C as competent cells.

The pET-28a(+) construct carrying CDK6 was transformed into E. coli BL21 (DE3) by heat shock. Plasmid DNA (50–100 ng) was mixed with chemically competent cells, incubated on ice for 20 minutes, exposed to 42 °C for 90 seconds, then returned to ice for 5 minutes before recovery in LB broth and plating on kanamycin-containing LB agar.

The full-length CDK6 gene (981 nucleotides) was cloned into pET-28a(+) and sequence-verified. Protein expression was carried out in E. coli BL21 (DE3), with induction by 0.25 mM IPTG.

Cells were lysed in buffer containing 25 mM Tris–HCl (pH 8.0), 200 mM NaCl, 1 mM DTT, and 10 mM PMSF, followed by sonication on ice. After centrifugation, the inclusion-body pellet was washed, solubilized in 25 mM Tris–HCl, 200 mM NaCl, and 0.5% sarcosine, and purified on a pre-equilibrated Ni–NTA column. Recombinant CDK6 was eluted with 150 mM imidazole and assessed by 12% SDS–PAGE.

Limonene inhibition of recombinant CDK6 was evaluated by a malachite green-based ATPase assay. Reaction mixtures contained purified CDK6 and freshly prepared ATP, and increasing concentrations of limonene were used to test dose dependence.

After incubation, malachite green reagent was added to quantify released phosphate, and absorbance was measured at 600 nm in a 96-well microplate format.

Intrinsic fluorescence spectroscopy was used to monitor limonene interaction with purified recombinant CDK6. Measurements were made on a JASCO spectrofluorometer (Model FP-6200), and titrations were performed with increasing limonene concentrations.

Fluorescence data were analyzed using Stern–Volmer and modified Stern–Volmer equations to derive the binding constant, number of binding sites, and thermodynamic implications of quenching.

Docking of limonene against CDK6 (3NUP) gave a predicted binding free energy of −6.3 kcal mol−1. The calculated pKi was 4.62 and the ligand efficiency was 0.63 kcal mol−1 per non-hydrogen atom.

The article states that the best-ranked docking pose occupies the active-site cavity and forms stabilizing interactions with key amino acid residues. Figure 3 presents the overall binding mode and a close-up view of the active-site pocket, while Figure 4 expands the analysis into 3D, 2D interaction-map, and surface-complementarity views.

Plasmid prepared with the QIAprep Spin Miniprep Kit showed purity of about 1.8 by A260/280 measurement. Agarose gel analysis showed nicked, linear, and supercoiled DNA forms, and the verified pET28a+ plasmid was successfully transformed into E. coli BL21 (DE3).

Colonies appeared on kanamycin plates after 12–16 hours at 37 °C. Recombinant CDK6 expression was induced with 0.25 mM IPTG, and purified protein was obtained through Ni–NTA affinity chromatography and confirmed by SDS–PAGE.

Progressive addition of limonene decreased the intrinsic fluorescence intensity of recombinant CDK6, which the authors interpret as quenching caused by ligand binding and changes in the local environment of aromatic fluorophores, mainly tryptophan residues.

The Stern–Volmer analysis supported concentration-dependent quenching and protein–ligand complex formation. The modified Stern–Volmer analysis was used to extract binding parameters, and the paper reports a strong limonene–CDK6 interaction.

Increasing limonene concentrations caused progressive reduction in CDK6 enzymatic activity, supporting dose-dependent suppression of kinase function.

The article links this functional inhibition to the docking and fluorescence findings, arguing that the limonene–CDK6 interaction is not merely computationally predicted but also biophysically and enzymatically supported. Figure 7 shows the decreasing enzyme activity trend versus ligand concentration.

This work received no funding.

Zulfareen acknowledges Jamia Millia Islamia for providing the Non-NET fellowship.

The authors declare no conflicts of interest.

All data generated or analyzed during this study are included in this manuscript.

The authors declare that ChatGPT (OpenAI) was used only to refine language, improve grammar, and enhance clarity of the manuscript.

The article explicitly states that AI tools were not used to generate scientific content, analyze data, interpret results, or draw scientific conclusions.