Molecular recognition of two bioactive coumarin derivatives 7-hydroxycoumarin and 4-methyl-7-hydroxycoumarin by hen egg white lysozyme: Exploring the binding mechanism, thermodynamic parameters and structural changes using multispectroscopic and computational approaches

Abstract

Multispectroscopic and computational methods of exploring the interaction between a carrier protein and therapeutic compounds provide a preliminary investigation into establishing the efficacy of such compounds. Here, two coumarin derivatives, 7-hydroxycoumarin (7-HC) and 4-methyl-7-hydroxycoumarin (4-Me-7-HC), were selected to carry out numerous biophysical interaction studies with a model carrier protein, hen egg white lysozyme (HEWL). Fluorescence spectroscopy studies conducted between HEWL and 7-HC/4-Me-7-HC revealed the binding constants (K_b) were in the range of 10⁴ M⁻¹, indicating a moderate nature of binding. The quenching mechanism observed during complexation process was an unusual static quenching due to the effect of temperature on the rate constant. Thermodynamic parameters revealed a positive ΔH and ΔS for HEWL-7-HC/4-Me-7-HC, indicating hydrophobic forces played a dominant role in the interaction process. FRET studies suggested a possible non-radiative energy transfer from the donor (HEWL) to the acceptor (coumarins). Molecular docking studies revealed the interaction of 7-HC/4-Me-7-HC with intrinsic fluorophores, Trp63 and Trp108, Trp108 being an essential residue for binding as proven by molecular dynamic (MD) simulation. MD simulation studies also indicated conformational stability gained by HEWL upon interaction with 7-HC and 4-Me-7-HC. The microenvironment surrounding the Trp residues showed a significant Stoke’s shift on carrying out 3-D fluorescence. CD studies revealed a decrease in the alpha helical content of HEWL upon interacting with the ligands. Enzymatic assay conducted for HEWL in the presence of 7-HC/4-Me-7-HC saw an increase in the activity of HEWL, suggesting a change in structural conformation and stability of the protein, altering its activity.

Communicated by Ramaswamy H. Sarma

Keywords:

• Hen egg white lysozyme
• 7-hydroxycoumarin
• 4-methyl-7-hydroxycoumarin
• fluorescence
• quenching
• docking

Acknowledgements

ASR is thankful to Science and Engineering Research Board (SERB) (Gov’t of India, File No. CRG/2019/000852) for funding.The authors are grateful to the Department of Bioscience and Bioengineering, IIT Guwahati for CD measurements. SL is grateful to St. Anthony’s College for enabling her to carry out her research work. The MD simulation was carried out by using the computational facility created under the grant no. SB/FT/CS-065/2012 and EMR/2017/001325. RG thanks NIT Rourkela for providing a scholarship.

Disclosure statement

No potential conflict of interest was reported by the authors.

Authors contribution

Sona Lyndem: Conceptualization, Investigation, Writing-Original draft

Rabiul Gazi: Software, Formal analysis, Writing-Original draft.

Vinay Kumar Belwal: Investigation

Madhurima Jana: Supervision, Software, Validation, Writing-Review & Editing, Funding acquisition.

Atanu Singha Roy: Supervision, Project administration, Validation, Writing - Review & Editing, Funding acquisition.