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

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 Förster 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-res

SUBMITTER: Heckmeier PJ 

PROVIDER: S-EPMC7335908 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature