On the Origin of the Extended Stoke's Shifts in Fluorescent Proteins

Abstract

Fluorescent proteins (FPs) emitting beyond 650 nm wavelengths are valuable for in vivo deep-tissue imaging due to their lower autofluorescence background, lower light scattering, and higher transmission compared to FPs emitting shorter wavelengths. Through molecular dynamics (MD) simulations of several mPlum variants, we showed that the dynamic Stoke's shift is correlated to the hydrogen bond switching around the the N-acylimine position of the extended chromophore. Specifically, the E16-I65 interaction to switch between direct and water-mediated h-bond states in mPlum was shown to be important for the large Stoke's shift. Recent QM/MM calculations have supported this observation by showing that the ground state population of the direct and water-mediated states shifts toward water-mediated state in the excited state. This occurs due to the redistribution of charges upon excitation, giving increased anionic characteristic to the acylimine oxygen which prefers the water-mediated h-bond. Classical MD show similar results of h-bond switching in TagRFP675 variants. TagRFP675 has the highest Stoke's shift of 77 nm and has hydrogen bond-network involving Q41 and S28 at the N-acylimine position. Rearrangement of the water molecule near acylimine moiety in TagRFP675 and mKate-M41Q allows extended hydrogen bond network connecting Q106-water-F65-Q41-S28. We have demonstrated this h-bond switching phenomenon in variants yet another FP family: eqFP650 and eqFP670. Compared to eqFP650, the rearrangement of the water molecule in eqFP670 gives a significantly extended h-bond network. All these examples highlight the importance of the fluidity of the water and other residues around acylimine environment for large Stokes shifts.