Insights on Aggregation of Hen Egg-White Lysozyme from Raman Spectroscopy and MD Simulations

Abstract

Protein misfolding and aggregation play a significant role in several neurodegenerative diseases. In the present work, the spontaneous aggregation of hen egg-white lysozyme (HEWL) in an alkaline pH 12.2 at an ambient temperature was studied to obtain molecular insights. The time-dependent changes in spectral peaks indicated the formation of β sheets and their effects on the backbone and amino acids during the aggregation process. Introducing iodoacetamide revealed the crucial role of intermolecular disulphide bonds amidst monomers in the aggregation process. These findings were corroborated by Molecular Dynamics (MD) simulations and protein-docking studies. MD simulations helped establish and visualize the unfolding of the proteins when exposed to an alkaline pH. Protein docking revealed a preferential dimer formation between the HEWL monomers at pH 12.2 compared with the neutral pH. The combination of Raman spectroscopy and MD simulations is a powerful tool to study protein aggregation mechanisms.

Keywords: Raman spectroscopy; molecular dynamics; protein aggregation; protein–protein interactions; structural biology.

Figure 1

(a) Optical image of the rings formed when the HEWL sample was drop-casted onto a hydrophobic glass substrate and dried in a desiccator; (b) 10× magnified image of the outer ring of the drop, the site from where the Raman spectrum was collected; (c) Raman spectra collected from the lyophilized HEWL monomer (red), rings of the 120 μM HEWL in pH 7.0 (green), and pH 12.2 (blue) at 0 h of the experiment.

Figure 2

Intensity of 505 cm⁻¹ Raman peak in (a) pH 7.0 and (b) pH 12.2; (c) intensity vs. time plot for 505 cm⁻¹ Raman peak; (d) various frames of the HEWL monomer at different simulation time points, in N and HBB conditions; (e) RMSD of the HEWL monomer in neutral pH and high pH with disulphide bonds broken in a 50 ns simulation; (f) secondary structure content in N and HBB after the simulation.

Figure 3

Stack plot of the Amide I Raman band of HEWL in (a) pH 7.0 and (b) pH 12.2; % α-helical and β-sheet content of the HEWL protein incubated in (c) pH 7.0 and (d) pH 12.2; (e) stack plot with Lorentzian fitting of the Amide III Raman band at selected time points for HEWL incubated in pH 12.2; (f) stack plot of Amide I Raman band of HEWL incubated in pH 12.2, with iodoacetamide added to it. Stack plot of the 1341 cm⁻¹ and 1363 cm⁻¹ doublet peaks of HEWL in (g) pH 7.0, (h) pH 12.2, and (i) pH 12.2 with iodoacetamide.

Figure 4

(a) Various kinds of interactions amidst the monomers N and HBB; (b) binding free energy of the docked proteins with the monomers N and HBB; (c) the total number of interactions in the dimers of the N and HBB systems; (d) a visual representation of the docked protein dimers in N and HBB indicating a more significant number of interactions in HBB as compared with N.