Encapsulation into complex coacervate core micelles promotes EGFP dimerization

Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer poly(2-methyl-vinyl-pyridinium)₄₁-b-poly(ethylene-oxide)₂₀₅. This packaging resulted in deviating spectroscopic features of the encapsulated EGFP molecules. Here we show that for monomeric EGFP variant (mEGFP) micellar encapsulation affects the absorption and fluorescence properties to a much lesser extent, and that changes in circular dichroism characteristics are specific for encapsulated EGFP. Time-resolved fluorescence anisotropy of encapsulated (m)EGFP established the occurrence of homo-FRET (Förster resonance energy transfer) with larger transfer correlation times in the case of EGFP. Together, these findings support that EGFP dimerizes whereas the mEGFP mainly remains as a monomer in the densely packed C3Ms. We propose that dimerization of encapsulated EGFP causes a reorientation of Glu222, resulting in a pK_a shift of the chromophore, which is fully reversible after release of EGFP from the C3Ms at a high ionic strength.