Excited State Vibrational Dynamics Reveals a Photocycle That Enhances the Photostability of the TagRFP-T Fluorescent Protein

High photostability is a desirable property of fluorescent proteins (FPs) for imaging, yet its molecular basis is poorly understood. We performed ultrafast spectroscopy on TagRFP and its 9-fold more photostable variant TagRFP-T (TagRFP S158T) to characterize their initial photoreactions. We find significant differences in their electronic and vibrational dynamics, including faster excited-state proton transfer and transient changes in the frequency of the v₅₂₀ mode in the excited electronic state of TagRFP-T. The frequency of v₅₂₀, which is sensitive to chromophore planarity, downshifts within 0.58 ps and recovers within 0.87 ps. This vibrational mode modulates the distance from the chromophore phenoxy to the side chain of residue N143, which we suggest can trigger cis/trans photoisomerization. In TagRFP, the dynamics of v₅₂₀ is missing, and this FP therefore lacks an important channel for chromophore isomerization. These dynamics are likely to be a key mechanism differentiating the photostability of the two FPs.