Oligomerisation, Fibrillation and Activity of Hen Lysozyme in Alkaline Medium: A Concentration Dependent Investigation

Abstract

Oligomerisation and fibrillation are hallmarks of protein misfolding diseases like Alzheimer's, Prion, Parkinson's, systemic amyloidosis and so on. Hen eggwhite lysozyme (HEWL) which spontaneously aggregates at pH 12.2 under room temperature as shown by us previously is an excellent model protein for aggregation studies. The present work was focussed on investigating the role of protein monomer concentration on the size and morphology of aggregates. For this purpose we employed HEWL concentrations ranging from 120 μM to 300 nM. Our findings reveal that A) FRET efficiency between dansyl labelled HEWL and dabcyl labelled HEWL in the aggregate, monitored over 12 hours was inversely proportional to HEWL monomer concentration B) Size exclusion chromatography after 12 hours showed that while all aggregates of 50 & 120 uM HEWL eluted much later, a small population of aggregates from 10 uM HEWL eluted early through the void volume C) Binding of ANS revealed fluorescence spectra that were gradually blue shifted and more intense, over 12h, in the following order with aggregates of 120 > 50 > 3 uM, while for 300 nM, these spectra were fairly invariant in emission wavelength and intensity over 12 hours. D) HEWL enzymatic activity decreased almost uniformly with time in alkaline pH for all concentrations suggesting a concentration independent early unfolding step E) AFM images showed extensive fibrils in 3 & 0.3 μM HEWL within 12 hours but predominantly large globular aggregates with 120 μM and 50 μM HEWL samples in the same time period. The above results clearly suggest that size and morphology of HEWL aggregates at alkaline pH are critically dependent on the initial monomer concentration.