Determining the structural origin of quantum behaviors in fluorescent proteins.

Abstract

Under physiological conditions fluorescent proteins (FP) in close proximity unexpectedly exhibit photophysical effects that are consistent with excitonic coupling: (1) ultrafast drop in anisotropy, (2) Davydov splitting in its CD spectra and (3) multiple FPs behaving as a single quantum emitter in photon antibunching experiments. We hypothesize that the FP β-barrel structure protects its internal chromophore from environmental decoherence, and thus slows its dephasing time relative to its energy transfer rate. To test this idea, we follow two parallel lines of investigation, one empirical and one theoretical. Experimentally, we prepare a catalog of fluorescent protein constructs that exhibit different combinations of energy transfer phenomena, namely excitonic coupling, and homo-FRET, and we characterize these using the three techniques mentioned above. The current list of FPs consists of Venus, LanYFP, Cerulean, EGFP, Tomato, and Ametrine, all of which were engineered into tandem dimer constructs and their appropriate monomeric controls. The variety of structurally related FP constructs should allow us to parse the origin of excitonic coupling, whether in the specific local structure along the β-barrel or the chromophore. In parallel, we employ molecular dynamics simulations to predict how the environment (the protein shell and/or solvent) impacts the excitation of the internal chromophore. We do this by estimating the total spectral density for excitation energy correlation functions as well as the contributions of composite structures to that spectral density. By applying this analysis on the various FP tandem dimer constructs, we can compare to what extent environmental coupling is predictive of observed quantum behaviors, and thus elucidate the role of structure.