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Heckmeier, P.J.* ; Agam, G.* ; Teese, M.G.* ; Hoyer, M.* ; Stehle, R. ; Lamb, D.C.* ; Langosch, D.*

Determining the stoichiometry of small protein oligomers using steady-state fluorescence anisotropy.

Biophys. J. 119, 99-114 (2020)

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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.

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