Title
Unravelling the Binding Mechanism and Protein Stability of Human Serum Albumin while Interacting with Nefopam Analogues: A Biophysical and Insilco approach
Author
Mahesh Gokara, Vidadala V. Narayana, Vineet Sadarangani, Shatabdi Roy Chowdhury, Sreelaxmi Varkala, Dhevalapally B. Ramachary, Rajagopal Subramanyam
Year
2016
Journal
Journal of Biomolecular Structure and Dynamics
Abstract
In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in the blood. Its binding affinity and concomitant changes in its conformation, binding site and simulations were also studied. Fluorescence data revealed that the fluorescence quenching of HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs binding constants (KA) are in the order of NF3>NF2>NF1 with values of 1.53 ± 0.057 × 104, 2.16 ± 0.071 ×104 and 3.6 ± 0.102 ×105 M−1, respectively. Concurrently, thermodynamic parameters indicate that the binding process was spontaneous, and the complexes were stabilized mostly by hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with hydrophobic interactions. Circular dichroism studies revealed that there is a decrease in α-helix with an increase in β-sheets and random coils signifying partial unfolding of the protein upon binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular docking studies showed that NF1, NF2 and NF3 bound to subdomain IIIA, IB and IIA through hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with LYS 413. Furthermore, molecular simulations unveiled that NF’s binding was in support with the structural perturbation observed in circular dichroism, which is evident from the RMSD and Rg fluctuations. We hope our insights will provide ample scope for engineering new drugs based on the resemblances with NFs for enhanced efficacy with HSA.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Ligand binding, Biochemistry