Decrease in Size of Hen Egg white Lysozyme Aggregates with Decrease in Monomer Concentration from Micro to Nanomolar in Alkaline pH

Abstract

Earlier work from our laboratory has demonstrated that hen egg white lysozyme (HEWL) is an excellent model protein for investigating protein aggregation at an alkaline pH of 12.2. Exposure of HEWL to this pH at monomer concentrations from 0.3 to 120 uM, yielded amyloid fibrils as shown previously. Here we present the following concentration dependent changes observed with HEWL aggregates subsequent to incubation at pH 12.2 at room temperature from 0 to 24 hours. A) a two-fold increase in exposure of trp residues in HEWL to water at 0.3 uM compared to 120 uM as revealed by fluorescence quenching experiments with iodide B) a several fold increase in ANS fluorescence intensity accompanied by significantly blue-shifted emission on binding to HEWL moving from 0.3 to 120 uM. C) a marginal increase in dansyl probe fluorescence intensity accompanied by significant blue-shifted dansyl emission in dansyl conjugated HEWL moving from 0.3 to 120 uM. D) a gradual increase in steady state fluorescence anisotropy of dansyl probe in dansyl conjugated HEWL moving from 3 to 120 uM, although the dansyl mean fluorescence lifetime remained concentration independent between 3 and 120 uM. E) presence of all cys in -SH form with absence of any change in free [-SH] between 20 & 120 uM, as revealed by DTNP assays. In addition, FCS measurements with rhodamine conjugated HEWL demonstrated facile aggregation of HEWL in the concentration range 25 nM to 120 uM. The above results imply that, the size of HEWL aggregates, are clearly dependent on the initial monomer concentration, with lower concentrations like 300 nM favoring small aggregates that possess solvent exposed trp, diminished binding towards ANS and shorter mean fluorescence lifetime for dansyl probe.