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.
Highlights
Cellular systems maintain the balance between protein synthesis and degradation via the quality-control machinery that prevents deposition of partially folded, misfolded or degraded protein in the cells [1]
Kinetics of Hen egg white lysozyme (HEWL) oligomerization process monitored by Thioflavin T (ThT) binding
To monitor the kinetics of HEWL aggregation, we undertook in vitro aggregation measurements in the presence of the ThT dye; the fluorescent properties of which change upon its binding to amyloid fibrils of proteins [46]
Summary
Cellular systems maintain the balance between protein synthesis and degradation via the quality-control machinery that prevents deposition of partially folded, misfolded or degraded protein in the cells [1]. A deregulation of these systems (i.e. change in cellular environment, genetic mutations, aging) can lead to various human diseases such as neurological disorders (i.e. Alzheimer's and Parkinson's diseases) and various systematic amyloidosis [2] These degenerative diseases are characterized by the formation and deposition of amyloid fibrils in different tissues and organs, resulting from uncontrolled self-aggregation of unfolded proteins [3,4,5]. Living organisms exploited the formation of amyloid fibrils as mechanism to perform physiological functions in specific biological contexts [6,7] These amyloid fibrils can act as functional bacterial coatings [8], natural protective systems against predators for various species [9], catalytic template in mammalian skin pigmentation [10] or as natural storage for peptides and proteins in secretory vesicles [11]. The formation of such protein amyloids is tightly controlled and regulated by cells whereas the aggregation of proteins in the degenerative diseases refers to their abnormal self-association
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