Comparative Study of Flavins Binding with Human Serum Albumin: A Fluorometric, Thermodynamic, and Molecular Dynamics Approach

Abstract

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derivatives of riboflavin (RF), a water-soluble vitamin, more commonly known as vitamin B(2). Flavins have attracted special attention in the last few years because of the recent discovery of a large number of flavoproteins. In this work, these flavins are used as extrinsic fluorescence markers for probing the microheterogeneous environment of a well-known transport protein, human serum albumin (HSA). Steady-state and time-resolved fluorescence experiments confirm that both FMN and FAD bind to the Sudlow's site-1 (SS1) binding pocket of HSA, where Trp214 resides. In the case of RF, a fraction of RF molecules binds at the SS1, whereas the major fraction of RF molecules remains unbound or surface bound to the protein. Moreover, flavin(s)-HSA interactions are monitored with the help of isothermal titration calorimetry, which provides free energy, enthalpy, and entropy changes of binding along with the binding constants. The molecular picture of binding interaction between flavins and HSA is well explored by docking and molecular dynamics studies.

Role of Mg2+ ions in flavin recognition by RNA aptamer

“…S 2 (k abs $ 375 nm) transitions ( Fig. 1) [27,32,[34][35][36]. With increasing concentration of RNA aptamer, absorption at both of $375 nm and $450 nm bands of FAD decreases, which becomes more prominent at higher Mg 2+ concentrations.…”

“…3a). Anisotropy dictates the extent of depolarization mainly contributed by the rotational diffusion process [27,32]. The molecular association process restricts the rotational diffusion of the fluorophore, and thereby leads to an increase in anisotropy.…”

“…Mg 2+ concentration dependent decrease in absorbance along with a red shift are preliminary indications toward the interaction between flavins and aptamer. Flavins (FAD/FMN) exhibit an unstructured emission at $530 nm in buffer (PBS, pH 7.2), which originates from isoalloxazine ring [26,27,32,35,36,38]. To explore the effect over flavin fluorescence upon binding to aptamer, a fixed concentration of FAD/ FMN in buffer is titrated by rising concentration of RNA aptamer (with 10 mM Mg 2+ ) (Fig.…”

“…Fluorescence lifetimes were collected from time-correlated single photon counting (TCSPC) set-up from IBH Horiba JobinYvon (U.S.A.) using 444 nm diode laser. The detail description of the instrument is mentioned elsewhere [32,33]. Analysis of lifetime data was done by IBH DAS6 software.…”

Characterization of zigzag patterns on the surface of bovine serum albumin films

“…Average segment lengths (L mean ) and angles between segments (α mean ) practically do not change. According to the studies [34][35][36], HSA and BSA form complexes with riboflavin (RF), flavin adenine dinucleotide (FAD) and FMN at a ratio of 1:1 and with high binding constants; in [36] it was also shown that, in the presence of RF, BSA conformation changes and its hydration increases. This agrees with the results obtained by spectroscopy and microwave dielectrometry for solutions of FMN with BSA [23].…”