Fluorophore Concentration Controls the Homo-FRET Efficiency and Directionality across Proteins both in Solution and in Solid Protein Matrices

Light harvesting in nature is one of the most important processes, starting with the absorption of light by chromophores within proteins and the transfer of energy from one another via the Förster resonance energy transfer (FRET) mechanism. If the energy is transferred between identical chromophores, the resulting is Homo-FRET. Here, we introduce an artificial system that allows us to control the number of chromophores within a certain protein and to decipher the mechanism of Homo-FRET in proteins in ways not possible in biological systems. We follow Homo-FRET by ultrafast fluorescence anisotropy measurements. We show that for solvated proteins, the mechanism, rate, and efficiency of Homo-FRET are highly dependent on the number of chromophores within the protein. However, in the solid protein matrix, the mechanism remains the same as the number of chromophores increases, but the solid matrix allows long-range Homo-FRET, resulting in full depolarization. The observed higher-than-ideal Homo-FRET contribution to the observed fluorescence anisotropy decay together with the reduction in the limiting anisotropy suggest a mixed coherent-incoherent mechanism of energy transfer.