title: Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay.
abstract: Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E (BoNT/A and BoNT/E). As detailed in that article, the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein (BFP) and green fluorescent protein (GFP) moieties linked via residues 134-206 of SNAP-25 (synaptosome-associated protein of 25kDa), the protein substrate for BoNT/A and BoNT/E. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is very low due to depolarization following energy transfer from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance, the polarization is high due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows an assay, termed DARET (depolarization after resonance energy transfer), that is robust and sensitive. In this article, we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarizations, and lifetimes.
authors.affiliation: Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, USA.
authors.forename: Justin A
authors.initials: JA
authors.lastname: Ross
authors.forename: Marcella A
authors.initials: MA
authors.lastname: Gilmore
authors.forename: Dudley
authors.initials: D
authors.lastname: Williams
authors.forename: K Roger
authors.initials: KR
authors.lastname: Aoki
authors.forename: Lance E
authors.initials: LE
authors.lastname: Steward
authors.forename: David M
authors.initials: DM
authors.lastname: Jameson
mesh_terms: D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011789:Quantum Theory; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E