title: Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis.
abstract: Characterization of the amyloidogenic Parkinson's disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled αS were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.
authors.affiliation: Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA. ejpetersson@sas.upenn.edu.
authors.forename: Conor M
authors.initials: CM
authors.lastname: Haney
authors.affiliation: Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA. ejpetersson@sas.upenn.edu.
authors.forename: Rebecca F
authors.initials: RF
authors.lastname: Wissner
authors.affiliation: Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA. ejpetersson@sas.upenn.edu.
authors.forename: John B
authors.initials: JB
authors.lastname: Warner
authors.affiliation: Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA. ejpetersson@sas.upenn.edu.
authors.forename: Yanxin J
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA. ejpetersson@sas.upenn.edu.
authors.forename: John J
authors.initials: JJ
authors.lastname: Ferrie
authors.affiliation: Center for Neurodegenerative Disease Research, University of Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104, USA.
authors.forename: Dustin
authors.initials: D
authors.lastname: J Covell
authors.affiliation: Center for Neurodegenerative Disease Research, University of Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Karpowicz
authors.affiliation: Center for Neurodegenerative Disease Research, University of Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104, USA.
authors.forename: Virginia M-Y
authors.initials: VM
authors.lastname: Lee
authors.affiliation: Department of Chemistry, University of Pennsylvania, 213 South 34th Street, Philadelphia, PA 19104, USA. ejpetersson@sas.upenn.edu.
authors.forename: E James
authors.initials: EJ
authors.lastname: Petersson
mesh_terms: D000682:Amyloid / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D013194:Staining and Labeling / Q000379:methods*; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000682:Amyloid; D051844:alpha-Synuclein