Ground and excited state proton transfer of the bioactive plant flavonol robinetin in a protein environment: spectroscopic and molecular modeling studies.

We performed spectroscopic and molecular modeling studies to explore the interaction of the bioactive plant flavonol robinetin (3,7,3',4',5'-OH flavone), with the carrier protein human serum albumin (HSA). Multiparametric fluorescence sensing, exploiting the intrinsic "two color" fluorescence of robinetin (comprising excited state intramolecular proton transfer (ESIPT) and charge transfer (CT) emissions) reveals that binding to HSA significantly affects the emission and excitation profiles, with strongly blue-shifted (∼29 nm) normal fluorescence and remarkable increase in the ESIPT fluorescence anisotropy (r) and lifetime (τ). Flavonol-induced HSA (tryptophan) fluorescence quenching data yield the dynamic quenching constant (KD) as 5.42 × 10(3) M(-1) and the association constant (Ks) as 5.59 × 10(4) M(-1). Time-resolved fluorescence anisotropy decay studies show dramatic (∼170 times) increase in the rotational correlation time (τ(rot)), reflecting greatly enhanced restrictions in motion of robinetin in the protein matrix. Furthermore, prominent induced circular dichroism (ICD) bands appear, indicating that the chiral environment of HSA strongly perturbs the electronic transitions of the intrinsically achiral robinetin molecule. Molecular docking calculations suggest that robinetin binds in subdomain IIA of HSA, where specific interactions with basic residues promote ground state proton abstraction and stabilize an anionic species, which is consistent with spectroscopic observations.

Interactions of dietary flavonoids with proteins: insights from fluorescence spectroscopy and other related biophysical studies.

In 1936, Rusznyak and Szent-Györgyi first drew attention to the therapeutically beneficial role of dietary flavonoids, which are the most common group of polyphenols ubiquitously present in plant based food and beverages. Recent years have witnessed a renascence of interest on these nutraceuticals, which, because of their high potency and low systemic toxicity, are gradually emerging as promising alternatives to conventional therapeutic drugs. There is a mounting evidence that various proteins frequently serve as targets for therapeutically important flavonoids. In this article we present perspectives exemplifying the growing potential of fluorescence spectroscopy as an exquisitely sensitive tool for noninvasive sensing of protein-flavonoid interactions at physiologically relevant concentrations, via measurements of steady state emission parameters as well as decay kinetics studies of the intrinsic fluorescence of the target (protein) and/or ligand (flavonoid). Especially, we highlight novel applications of the remarkably environment sensitive 'two color' fluorescence exhibited by many important flavonoids, which permits multiparametric and ratiometric measurements. To consolidate findings obtained via fluorescence spectroscopy, use of other relevant experimental biophysical techniques and molecular modeling have proved to be valuable and are also discussed here. Such complementary studies provide additional insights regarding the thermodynamics and conformational aspects of the protein-flavonoid interactions, together with details, at atomistic level, of the dominant noncovalent interactions involved in the docking of different flavonoids to their target proteins.