A common steric effect controlling fluorescence lifetime in jellyfish- and coral-derived fluorescent proteins

Fluorescent proteins (FPs) are widely used as genetically encoded probes for visualizing and analyzing biological processes. Fluorescence lifetime is an important photophysical property of FPs, yet a general principle governing lifetime across different FPs remains unclear. We previously showed that the fluorescence lifetimes of the Aequorea victoria-derived FPs enhanced yellow fluorescent protein and enhanced green fluorescent protein depend on the side chain volume at residue 145, and that this relationship also accounts for reported lifetime differences among other Aequorea victoria-derived FPs. Here, this relationship was examined in the coral-derived red FPs mOrange, mStrawberry, and mCherry. Trp143 was identified as the corresponding residue and replaced by each of the other 19 amino acids. In all three FPs, larger side chains at residue 143 generally produced longer fluorescence lifetimes, whereas smaller side chains produced shorter lifetimes, consistent with steric restriction of chromophore motion. The magnitude of lifetime modulation differed among the proteins: mOrange and mStrawberry showed large variations, whereas mCherry showed a smaller variation. Based on structural comparisons and reported molecular dynamics studies, the local environment around residue 163 is proposed to contribute to these differences. Together with previous results for Aequorea victoria-derived FPs, these findings suggest that the steric environment near the phenolate oxygen of the chromophore is a common factor controlling fluorescence lifetime through restriction of chromophore motion, while hydrogen-bonding interactions, local polarity, and packing modulate the magnitude of the response. This principle may guide the rational control of FP fluorescence lifetime and the development of FPs with improved photophysical properties.