Determining the Stoichiometry of Small Protein Oligomers Using Steady-State Fluorescence Anisotropy

Heckmeier, Philipp J.; Agam, Ganesh; Teese, Mark G.; Hoyer, Maria; Stehle, Ralf; Lamb, Don C. und Langosch, Dieter (2020): Determining the Stoichiometry of Small Protein Oligomers Using Steady-State Fluorescence Anisotropy. In: Biophysical Journal, Bd. 119, Nr. 1: S. 99-114

Abstract

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Forster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (k(FRET)), average fluorescence lifetimes (tau), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (r(ET)) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photo-bleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Chemie und Pharmazie > Department Chemie
• Fakultätsübergreifende Einrichtungen: Center for NanoScience (CENS)
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie ; 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
• ISSN: 0006-3495
• Sprache: Englisch