Effects of C-ring structures on the formations of flavonoid semiquinones and their binding behavior with β-lactoglobulin as revealed by experimental and modeling approaches

The present study investigated the covalent binding behavior of the flavonoids, catechin, eriodictyol, luteolin and quercetin with β-lactoglobulin (βlg). Since the four flavonoids possess the identical A- and B-ring structures, effects of the C-rings on the properties of flavonoids and the corresponding semiquinones are revealed. Experimental methods including DLS and CD spectra indicated that with quercetin at room temperature did not induce aggregation of βlg, whilst binding with the other three flavonoids resulted in aggregation of βlg. Proteomic and LC-MS/MS analysis indicated that lysine (Lys/K), tryptophan (Trp/W) and cysteine (Cys/C) exhibited high propensity to bind with flavonoids. Catechin exhibited the highest binding with βlg while eriodictyol had the lowest and showed no tendency to bind with tryptophan (Trp/W). Quantum chemistry calculations showed that the corresponding semiquinones with the deprotonations occurring on the A-rings exhibited higher reactivity than those with radicals on the B-rings. Behavior of flavonoid semiquinones formed after deprotonation influenced the protein stability as revealed by MD simulations. Assemblies of quercetin semiquinones were found to protect βlg from unfolding and aggregation. The modeling provided explanations for experimental observations, and provided new insights and understanding of covalent interactions between proteins and flavonoids.