(1) Background: Protein–polyphenol interactions have been widely studied regarding their influence on the properties of both protein and the ligands. As an important protein material in the food industry, soybean protein isolate (SPI) experiences interesting changes through polyphenols binding. (2) Methods: In this study, a molecular docking and virtual screening method was established to evaluate the SPI–polyphenol interaction. A compound library composed of 33 commonly found food source polyphenols was used in virtual screening. The binding capacity of top-ranking polyphenols (rutin, procyanidin, cyanidin chloride, quercetin) was validated and compared by fluorescence assays. (3) Results: Four out of five top-ranking polyphenols in virtual screening were flavonoids, while phenolic acids exhibit low binding capacity. Hydrogen bonding and hydrophobic interactions were found to be dominant interactions involved in soybean protein–polyphenol binding. Cyanidin chloride exhibited the highest apparent binding constant (Ka), which was followed by quercetin, procyanidin, and rutin. Unlike others, procyanidin addition perturbed a red shift of SPI fluorescence, indicating a slight conformational change of SPI. (4) Conclusions: These results suggest that the pattern of SPI–polyphenol interaction is highly dependent on the detailed structure of polyphenols, which have important implications in uncovering the binding mechanism of SPI–polyphenol interaction.

Soybean protein isolate (SPI) and small molecule interactions have drawn more and more attention regarding their benefits for both parts, while research on large-scale investigations and comparisons of different compounds is absent. In this study, a high throughput virtual screening was applied on a phytochemical database with 1130 compounds to pinpoint the potential SPI binder. Pentagalloylglucose, narcissoside, poliumoside, isoginkgetin, and avicurin were selected as the top-five ranking molecules for further validation. Fluorescence quenching assays illustrated that isoginkgetin has a significantly higher apparent binding constant (Ka) of (0.060 ± 0.020) × 10⁶ L·mol⁻¹, followed by avicularin ((0.058 ± 0.010) × 10⁶ L·mol⁻¹), pentagalloylglucose ((0.049 ± 0.010) × 10⁶ L·mol⁻¹), narcissoside ((0.0013 ± 0.0004) × 10⁶ L·mol⁻¹), and poliumoside ((0.0012 ± 0.0006) × 10⁶ L·mol⁻¹). Interface characterization by MD simulation showed that protein residues E172, H173, G202, and V204 are highly involved in hydrogen bonding with the two carbonyl oxygens of isoginketin, which could be the crucial events in SPI binding. Van der Waals force was identified as the major driven force for isoginketin binding. Our study explored SPI–phytochemical interaction through multiple strategies, revealing the molecular binding details of isoginkgetin as a novel SPI binder, which has important implications for the utilization of the SPI–phytochemical complex in food applications.

(1) Background: The mitochondrial permeability transition pore (mPTP) contributes to mitochondrial dysfunction under pathological conditions. Cyclophilin D (CypD), a key regulator of sustained mPTP opening, represents an important molecular target. However, systematic strategies for selecting an appropriate CypD crystal structure for virtual screening and efficiently identifying CypD-binding natural polyphenols remain limited. (2) Methods: Six CypD crystal structures were evaluated by receiver operating characteristic analysis using reported CypD inhibitors and approximately 4000 decoys. The optimal receptor structure was subsequently used to screen 484 dietary polyphenols by molecular docking. Selected top-ranked candidates were further characterized by ¹⁵N–¹H HSQC NMR titration and surface plasmon resonance (SPR). (3) Results: PDB 6R8L exhibited the best virtual-screening performance, particularly in early enrichment. Several polyphenol glucuronides ranked among the top candidates. NMR analysis showed that apigenin 7-glucuronide and luteolin 7-glucuronide induced pronounced chemical shift perturbations at CypD residues F60, M61, I117, and W121, with F60, M61, and W121 located within or near the docking-predicted S1 binding region. SPR independently confirmed direct binding, yielding K_D values of 66 μM and 84 μM for apigenin 7-glucuronide and luteolin 7-glucuronide, respectively. (4) Conclusions: Integrating systematic receptor evaluation, molecular docking, and complementary NMR and SPR characterization provides a practical workflow for identifying and characterizing natural CypD-binding compounds. Apigenin 7-glucuronide and luteolin 7-glucuronide were identified as micromolar CypD-binding candidates, highlighting polyphenol glucuronides as a chemical class warranting further investigation in CypD ligand discovery.