Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods

Abstract

Protein misfolding and amyloid formation are an underlying pathological hallmark in a number of prevalent diseases of protein aggregation ranging from Alzheimer’s and Parkinson’s diseases to systemic lysozyme amyloidosis. In this context, we have used complementary spectroscopic methods to undertake a systematic study of the self-assembly of hen egg-white lysozyme under agitation during a prolonged heating in acidic pH. The kinetics of lysozyme aggregation, monitored by Thioflavin T fluorescence, dynamic light scattering and the quenching of tryptophan fluorescence by acrylamide, is described by a sigmoid curve typical of a nucleation-dependent polymerization process. Nevertheless, we observe significant differences between the values deduced for the kinetic parameters (lag time and aggregation rate). The fibrillation process of lysozyme, as assessed by the attenuated total reflection-Fourier transform infrared spectroscopy, is accompanied by an increase in the β-sheet conformation at the expense of the α-helical conformation but the time-dependent variation of the content of these secondary structures does not evolve as a gradual transition. Moreover, the tryptophan fluorescence-monitored kinetics of lysozyme aggregation is described by three phases in which the temporal decrease of the tryptophan fluorescence quantum yield is of quasilinear nature. Finally, the generated lysozyme fibrils exhibit a typical amyloid morphology with various lengths (observed by atomic force microscopy) and contain exclusively the full-length protein (analyzed by highly performance liquid chromatography). Compared to the data obtained by other groups for the formation of lysozyme fibrils in acidic pH without agitation, this work provides new insights into the structural changes (local, secondary, oligomeric/fibrillar structures) undergone by the lysozyme during the agitation-induced formation of fibrils.

Probing the fibrillation of lysozyme by nanoscale-infrared spectroscopy

“…During the fibrillation of lysozyme, an increase in b-sheet content occurs gradually, as highlighted by CD as well as IR studies. The increase in b-sheet content is consistent with previous results on lysozyme by other groups (Chaari, Fahy, Chevillot-Biraud, & Rholam, 2015;Frare et al, 2009;Malisauskas et al, 2003) and indicates the common feature during amyloid fibrillation.…”

Amyloid fragments and their toxicity on neural cells

“…Ultrasonic treatment (UST) is often used for preparation of fibril seeds in studies of amyloid aggregation. It breaks fibrils into shorter pieces [7] increasing the number of fibril ends that act as active aggregation sites and accelerating elongation rate without a change of total protein concentration. Fragments of fibrils were obtained via sonication of mature fibril samples.…”

Molecular Insight into Human Lysozyme and Its Ability to Form Amyloid Fibrils in High Concentrations of Sodium Dodecyl Sulfate: A View from Molecular Dynamics Simulations

“…Systemic amyloidosis is a lysozyme-associated disease caused by the deposition of lysozyme in the amyloid fibril form in spleen, kidney, and liver cells [ 33 , 36 ]. Previous studies have shown that lysozyme can easily form amyloid fibrils under different conditions, such as high temperatures and appropriate pH conditions [ 37 – 40 ]. Many amyloidogenic proteins interact with the surfaces of basement membranes; SDS has the potential to mimic this condition [ 29 , 41 – 45 ].…”