title: Kaede for detection of protein oligomerization.
abstract: Photoconvertible fluorescent proteins such as Kaede are routinely used for tracking proteins, organelles, and whole cells. Kaede was the first identified photoconvertible fluorescent protein and has since become the most commonly used photoconvertible fluorescent protein in vertebrates. Kaede can be irreversibly converted from a green to a red fluorescent form upon UV/blue light irradiation and fluorescence of each form can be isolated separately by appropriate filter sets. Spectral properties of the Kaede forms allow Förster resonance energy transfer (FRET) from the green form as donor to the red form as acceptor. As a sample containing oligomerized Kaede-containing proteins is exposed to UV or blue light, FRET first increases as green Kaede is converted to red and then decreases as the green donor becomes depleted. Thus, FRET information is potentially obtained from a number of independent measurements taken as photoconversion proceeds. We demonstrate here the application of this approach to detect homo-aggregation and conformational dynamics of plant protein constructs. Structural alterations of 2-cys peroxiredoxin–Kaede were successfully detected depending on the redox state in living plant cells. Photoconversion was performed gradually and donor emission, acceptor emission, and FRET-derived sensitized acceptor emission were measured at each step of conversion. Since photoconvertible proteins have not been routinely used in plants, two plasmids have been designed to facilitate plant applications. The plasmids allow either transient expression of Kaede-containing protein constructs in plant cells or Gateway cloning and stable transformation of plants.
authors: Dynamic Cell Imaging, Faculty of Biology, Bielefeld University, D-33501 Bielefeld, Germany.
authors: Heike
authors: H
authors: Wolf
authors: B George
authors: BG
authors: Barisas
authors: Karl-Josef
authors: KJ
authors: Dietz
authors: Thorsten
authors: T
authors: Seidel
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D007834:Lasers; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism*; D055620:Optical Phenomena; D054464:Peroxiredoxins / Q000378:metabolism; D059828:Plant Cells / Q000378:metabolism / Q000528:radiation effects; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding / Q000528:radiation effects; D055503:Protein Multimerization; D055550:Protein Stability / Q000528:radiation effects; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000528:radiation effects
chemical_list: C513582:Kaede protein, Trachyphyllia geoffroyi; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054464:Peroxiredoxins
keywords: fluorescence imaging; protein structure; protein–protein interaction