Abstract
Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions.
Highlights
Since its discovery in the 1920’s, lysozyme has increasingly emerged as an important system for studying protein structure and function [1,2]
We have successfully introduced a site-specific biotin label into WT human lysozyme and have shown that this modification has no significant effects on native-state structure and stability or on the process of in vitro fibril formation by the biotinylated lysozyme
It has been demonstrated in previous studies that the regions of human lysozyme containing the bdomain and the C-helix in the native state are unfolded in the transiently partially unfolded intermediate which is crucial for fibril formation [24,65]
Summary
Since its discovery in the 1920’s, lysozyme has increasingly emerged as an important system for studying protein structure and function [1,2]. The human form of lysozyme is a globular protein containing 130 amino acids and its native structure is typical of the c-type lysozymes [6]. There have been many investigations of the folding mechanism of human lysozyme (see references within [7]) These studies became of particular interest following the discovery of two natural variants of lysozyme, I56T and D67H, which are linked to systemic amyloidosis. This fatal disorder is associated with the enhanced propensity of these naturally occurring mutational variants to self-assemble into amyloid fibrils [8]. Since the discovery of the I56T and D67H variants, four other naturally occurring disease-associated variants (F57I, F57I/T70N, W64R and T70N/W112R) have been identified, along with an additional variant (T70N) which is not disease-associated [13,14,15,16]
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