Investigating the Binding Interaction between β-Lactoglobulin and Vitamin B12: A Spectroscopic and Computational Approach

Abstract

Binding interaction of the major whey protein β-Lactoglobulin (βLG) with vitamin B12 is crucial for understanding the potency of βLG as a transporter for vitamin B12. Hence, the binding interaction of the βLG with vitamin B12 was studied using spectroscopic (UV-Visible absorption spectroscopy, steady-state & time-resolved fluorescence spectroscopy, synchronous fluorescence spectroscopy (SFS), circular dichroism (CD), and fluorescence correlation spectroscopy (FCS)) and computational (molecular docking and molecular dynamics (MD) simulation) tools. The quenching of the intrinsic fluorescence of βLG by vitamin B12 confirms the binding of vitamin B12 with βLG. The nature of the quenching is found to be static due to ground state complex formation. The calculated thermodynamic parameters suggest the binding process is hydrophobic. Synchronous fluorescence spectroscopy revealed that vitamin B12 affects the tryptophan residue microenvironment of the protein. Vitamin B12 does not alter the secondary and tertiary structure of βLG, as concluded from the CD and FCS experiments. From the molecular docking results, vitamin B12 is found to bind at the β- barrel site of βLG. The MD simulation results suggest that vitamin B12 bound βLG complex is stable. The secondary structural element analysis of MD simulation results show no alteration in protein secondary structure after binding with vitamin B12, which is matched with the experimental findings. This biophysical binding interaction study of βLG with vitamin B12 might have promising applications in the pharmaceutical and food industries.