The expression vector pET-28a(+) and Escherichia coli BL21 (DE3) competent cells were procured from Qiagen, while E. coli DH5$\alpha$ cells were obtained from Invitrogen. Analytical-grade reagents, including sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), and other routine chemicals, were purchased from Merck (India). Luria-Bertani (LB) broth was obtained from Merck (Darmstadt, Germany). Kanamycin and isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) were sourced from Sigma-Aldrich (St. Louis, MO, USA). Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography columns and Ni-NTA agarose beads were acquired from Bio-Rad and Qiagen (QIAexpress), respectively. Limonene used in this study was purchased from Sigma-Aldrich (USA).

Molecular docking was performed to investigate the binding interactions between Conformer3D_COMPOUND_CID_22311 (limonene) and the crystal structure of CDK6 (PDB ID: 3NUP). Docking simulations were conducted using InstaDock, an automated platform for structure-based virtual screening (Mohammad et al., 2021). Binding affinity calculations were carried out using QuickVina-W (Hassan et al., 2017), a modified version of AutoDock Vina (Trott and Olson, 2010) that integrates empirical and knowledge-based scoring functions. A blind docking approach was employed to allow unbiased exploration of potential ligand-binding sites across the protein surface. The predicted binding free energy ($\Delta G$, kcal mol^-1) obtained from docking was used to calculate the inhibitory constant ($K_{i,\mathrm{pred}}$) and $\mathrm{p}{K}_i$ values according to the following thermodynamic relationships:

$$\Delta G=RT\ln K_{i,\mathrm{pred}}$$

$$K_{i,\mathrm{pred}}=\exp \left(\frac{\Delta G}{RT}\right)$$

$$\mathrm{p}{K}_i=-{\log }_{10}{K}_{i,\mathrm{pred}}$$

where $\Delta G$ represents the docking-derived binding free energy, $R$ is the universal gas constant ($1.98\mathrm{cal}{\mathrm{mol}}^{-1}$ K^-1), $T$ is the temperature (298.15 K), and $K_{i,\mathrm{pred}}$ is the predicted inhibition constant (Shityakov, 2014).

To further evaluate ligand binding efficiency, ligand efficiency (LE) was calculated using:

$$\mathrm{LE}=-\frac{\Delta G}{N}$$

where LE denotes ligand efficiency (kcal mol^-1 per non-hydrogen atom), $\Delta G$ is the binding free energy, and $N$ corresponds to the number of non-hydrogen atoms in the ligand. This parameter provides a normalized assessment of binding strength relative to molecular size (Hopkins et al., 2004).

The overnight bacterial culture was pelleted by centrifugation at 6000 rpm for 5 minutes at room temperature, then resuspended in the resuspension buffer. Lysis was performed to release cellular contents, including plasmid DNA, using the lysis buffer. Proteins were then digested using proteinase K. Purification of the plasmid was done using a phenol-chloroform extraction kit. Finally, the concentration and purity of plasmid DNA were checked using a Nanodrop at 260 nm. Purified bands of isolated plasmid were also confirmed using agarose gel electrophoresis.

A 0.8% agarose gel was prepared by adding TAE buffer and heating in a microwave. EtBr was added to the cooled gel solution, and then the cooled gel was poured into the gel tray. Gel was allowed to solidify for about 20 min. Samples were prepared by adding loading dye (bromophenol blue) to the isolated plasmid DNA, and loading the samples into the wells after removing the comb. The tank was filled with TAE buffer, and the leads were connected to the power supply. After applying a constant voltage of 100-150 V for about 45-60 minutes, the gel was removed from the gel box, placed on a UV transilluminator, and visualized using a gel documentation system.

A single colony was inoculated into LB broth containing kanamycin and incubated overnight at 37 °C, then transferred to a larger volume of fresh LB broth and incubated until the OD reached 0.4-0.6 at 600 nm. Metabolic activity was reduced by cooling the culture on ice for 15-30 minutes. Culture was centrifuged, and the supernatant was discarded. Pellet was resuspended in ice-cold 10% glycerol solution. Competent cells formed was aliquoted in microcentrifuge tubes and stored at $-80$ °C (Bird et al., 2022).

The recombinant plasmid construct pET-28a(+) harboring the CDK6 gene was transformed into Escherichia coli BL21 (DE3) competent cells using the heat-shock method. Briefly, chemically competent BL21 (DE3) cells were thawed on ice, and 50-100 ng of plasmid DNA was gently mixed with the cells. The mixture was incubated on ice for 20 min to facilitate DNA adsorption. Subsequently, cells were subjected to a heat shock at 42 °C for 90 s, followed by immediate ice incubation for 5 min to stabilize the membranes. Thereafter, 900 $\mu$L of Luria-Bertani (LB) broth was added, and the cells were allowed to recover at 37 °C with shaking at 220 rpm for 1 h. The transformed cells were then spread onto LB agar plates supplemented with kanamycin and incubated overnight (12-16 h) at 37 °C to allow colony formation (Yousuf et al., 2022).

The full-length CDK6 gene (981 nucleotides) was cloned into the pET-28a(+) expression vector and sequence-verified prior to protein expression. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells for heterologous protein production. Transformed cells were cultured in LB medium containing kanamycin at 37 °C until the desired optical density was reached, then induced with 0.25 mM isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). After induction, cells were harvested by centrifugation and resuspended in lysis buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM DTT, and 10 mM PMSF). Cell disruption was performed by sonication on ice, and the lysate was centrifuged at 9,000 rpm for 20 min at 4 °C to separate soluble and insoluble fractions. The pellet containing inclusion bodies was collected and washed three times with Milli-Q water to remove impurities.

The inclusion bodies were subsequently solubilized in solubilization buffer (25 mM Tris-HCl, 200 mM NaCl, and 0.5% sarcosine) and loaded onto a pre-equilibrated Ni-NTA affinity chromatography column for purification. After binding, the column was washed to remove non-specifically bound proteins, and recombinant CDK6 was eluted using elution buffer containing 150 mM imidazole (25 mM Tris-HCl, pH 8.0, 200 mM NaCl). Protein purity and molecular weight were analyzed using 12% SDS-PAGE (Yousuf et al., 2020).

The inhibitory effect of limonene on recombinant CDK6 was evaluated using a malachite green-based ATPase assay. The reaction mixture (final volume: 50 $\mu$L) contained purified CDK6 (1 $\mu$M) and freshly prepared ATP (200 $\mu$M). Increasing concentrations of limonene were added to assess dose-dependent inhibition. The reaction mixtures were incubated at 37 °C for 45 min to allow ATP hydrolysis. The reaction was terminated by adding 100 $\mu$L of malachite green reagent, followed by incubation at room temperature for 15-20 min to enable color development resulting from inorganic phosphate release. Absorbance was measured at 600 nm using a microplate ELISA reader in a 96-well format. Enzyme activity was calculated based on phosphate release, and percentage inhibition was determined relative to control reactions lacking inhibitor (Voura et al., 2019).

The interaction between limonene and purified recombinant CDK6 was investigated using intrinsic fluorescence spectroscopy by monitoring changes in the emission spectrum of CDK6 upon ligand titration (Jameel et al., 2017). Fluorescence measurements were performed using a JASCO spectrofluorometer (Model FP-6200). Titration experiments were conducted by progressively increasing the limonene concentration, and each measurement was performed in triplicate to ensure reproducibility. Changes in fluorescence intensity were recorded and analyzed to determine binding parameters. The binding constant (Ka), number of binding sites ($n$), and associated thermodynamic parameters were calculated from fluorescence quenching data using the Stern-Volmer equation. Analysis of the quenching behavior enabled characterization of the binding affinity and interaction mechanism between limonene and CDK6 (Shamsi et al., 2020).

$$\frac{F_0}{F}=1+K_{\mathrm{SV}}[Q]=1+k_q{\tau }_0[Q]$$

$$\log \left(\frac{F_0-F}{F}\right)=\log K_a+n\log [Q]$$

Docking analysis of Conformer3D_COMPOUND_CID_22311 (limonene) against the CDK6 crystal structure (PDB ID: 3NUP) revealed a predicted binding free energy ($\Delta G$) of $-6.3\mathrm{kcal}{\mathrm{mol}}^{-1}$. The corresponding calculated $\mathrm{p}{K}_i$ value was 4.62, indicating moderate binding affinity toward the target protein. The ligand efficiency (LE) was determined to be $0.63\mathrm{kcal}{\mathrm{mol}}^{-1}$ per non-hydrogen atom, suggesting favorable binding energy relative to molecular size (Figure 3).

Comprehensive interaction profiling of all generated docked conformations was performed to examine the binding orientation and molecular interactions within the 3NUP binding pocket (Figure 3). Analysis of the best-ranked pose revealed that the ligand occupies the active-site cavity and establishes stabilizing interactions with key amino acid residues, thereby contributing to its predicted binding affinity.

Plasmid isolated using the QIAprep Spin Miniprep Kit was checked for purity and concentration using a Nanodrop, which showed a purity of about 1.8 ($A_{260}/A_{280}$), and digested bands were visualized by running an agarose gel, which showed clear bands for nicked, linear, and supercoiled forms of DNA (Figure 5A). The confirmed pET-28a(+) plasmid was successfully transformed into E. coli strain BL21 (DE3). Colonies appear after incubation with kanamycin at 50 $\mu$g/mL for 12-16 hours at 37 °C (Figure 5B). Recombinant CDK6 protein was expressed at 37 °C by inducing with 0.25 mM IPTG. Purified CDK6 protein was obtained by Ni-NTA affinity chromatography, confirmed by running SDS-PAGE (Figure 5C).

Intrinsic fluorescence spectroscopy was employed to evaluate the interaction between limonene and recombinant CDK6. Progressive addition of limonene led to a marked decrease in the intrinsic fluorescence intensity of CDK6, indicating effective quenching of the protein's fluorescence (Figure 6A). This observation suggests that ligand binding alters the local microenvironment surrounding aromatic fluorophores (primarily tryptophan residues), thereby affecting emission characteristics. The fluorescence quenching data were analyzed using both the Stern-Volmer and modified Stern-Volmer equations to determine the quenching mechanism and binding parameters. The Stern-Volmer plot (Figure 6B) demonstrated a concentration-dependent quenching profile, supporting the formation of a protein-ligand complex.

Binding parameters derived from the modified Stern-Volmer plot (Figure 6B) revealed a binding constant (Ka) of $4.1\times 10^7{\mathrm{M}}^{-1}$, indicative of strong affinity between limonene and CDK6. The slope of the modified plot further provided the number of binding sites ($n$), suggesting a defined binding interaction. The high binding constant obtained from fluorescence analysis is consistent with the molecular docking results, collectively supporting a stable and favorable interaction between limonene and CDK6.

The inhibitory effect of limonene on recombinant CDK6 kinase activity was evaluated in a dose-dependent manner. Increasing concentrations of limonene resulted in a progressive reduction in CDK6 enzymatic activity (Figure 7). The observed decrease in kinase activity demonstrates that limonene effectively suppresses CDK6 function, supporting its potential role as a kinase inhibitor. These findings are consistent with both the molecular docking predictions and fluorescence binding studies, collectively indicating a stable interaction that translates into functional inhibition of the enzyme.