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.