Different dimerization affinity and orientation of fluorescent proteins eGFP and eYFP

Oligomerization of fluorescent proteins (FPs) is critical for imaging but is poorly understood. We investigated the dimerization of enhanced green fluorescent protein (eGFP) and enhanced yellow fluorescent protein (eYFP), which differ by only five residues. Size exclusion chromatography (SEC) confirmed a monomer-dimer equilibrium for both eGFP and eYFP. Using analytical ultracentrifugation (AUC), the dissociation constant (K_d) was determined to be 340 µM for eGFP, indicating a significantly lower affinity than eYFP (K_d = 20 µM). Time-resolved fluorescence anisotropy revealed that intra-dimer homo-Förster resonance energy transfer (FRET) is completed faster than 20 ps for both FPs. Importantly, by analyzing concentration-dependent anisotropy signals, we independently determined that the K_d values are 740 µM for eGFP and 36 µM for eYFP, providing more reliable estimates for FPs with low affinity. Furthermore, we determined that the transition dipole moment angles within the homodimers are 32° for eGFP and 49° for eYFP. These findings demonstrate that subtle sequence variations can significantly alter both interfacial affinity and relative orientation. This study contributes to the rational design of FRET sensors by enabling the independent control of dimerization affinity and relative orientation.