title: Analysis of the native structure, stability and aggregation of biotinylated human lysozyme.
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.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Minkoo
authors.initials: M
authors.lastname: Ahn
authors.forename: Erwin
authors.initials: E
authors.lastname: De Genst
authors.forename: Gabriele S
authors.initials: GS
authors.lastname: Kaminski Schierle
authors.forename: Miklos
authors.initials: M
authors.lastname: Erdelyi
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.forename: Janet R
authors.initials: JR
authors.lastname: Kumita
mesh_terms: D000682:Amyloid / Q000096:biosynthesis*; D028226:Amyloidosis, Familial / Q000378:metabolism*; D001360:Avidin / Q000378:metabolism; D001703:Biophysics; D019921:Biotinylation; D002942:Circular Dichroism; D005818:Genetic Engineering; D006801:Humans; D015151:Immunoblotting; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000682:Amyloid; D001360:Avidin; D009113:Muramidase; C514728:lysozyme C, human