title: Towards multiparametric fluorescent imaging of amyloid formation: studies of a YFP model of alpha-synuclein aggregation.
abstract: Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein alpha-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.
authors.affiliation: Department of Genetics, University of Groningen, Groningen, The Netherlands.
authors.forename: Tjakko J
authors.initials: TJ
authors.lastname: van Ham
authors.forename: Alessandro
authors.initials: A
authors.lastname: Esposito
authors.forename: Janet R
authors.initials: JR
authors.lastname: Kumita
authors.forename: Shang-Te D
authors.initials: ST
authors.lastname: Hsu
authors.forename: Gabriele S
authors.initials: GS
authors.lastname: Kaminski Schierle
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.forename: Ellen A A
authors.initials: EA
authors.lastname: Nollen
authors.forename: Carlos W
authors.initials: CW
authors.lastname: Bertoncini
mesh_terms: D000682:Amyloid / Q000096:biosynthesis* / Q000737:chemistry* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D066298:In Vitro Techniques; D016631:Lewy Bodies / Q000378:metabolism / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D019906:Nuclear Magnetic Resonance, Biomolecular; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D000682:Amyloid; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051844:alpha-Synuclein; C054562:yellow fluorescent protein, Bacteria