Revealing Ultrafast Structural Motions of the GFP Model Chromophore in Solution

Fluorescent proteins as biomarkers have provided decades of valuable research insights for the scientific and engineering community. The green fluorescent protein (GFP) has been a favorite for bioimaging due to its bright fluorescence and high quantum yield. Its success has inspired bioengineers to modify the chromophoreâ??s structure and environment to create many new fluorescent proteins with better properties. Despite the large success of engineered GFP derivatives, ongoing research seeks to solve some of the drawbacks of current fluorescent proteins, including low brightness, fast photodegradation, and poor contrast. Critical to this research is knowledge about the interplay between chromophore fluorescence and local environment, a paradigm that is still not fully understood for GFP. Notably, the specific excited state structural motions preventing the GFP model chromophore 4-hydroxybenzylidene-1,2-dimethylimidazolinone (HBDI) from significantly fluorescing when taken out of the protein pocket are undetermined.

Femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with transient absorption (TA) spectroscopy provides critical insights into the structural motions that compete with fluorescence when HBDI is in solution. Analysis of intensity and frequency dynamics reveals that the molecule undergoes internal conversion by evolving into a twisted internal charge transfer (TICT) state on a ~2.0 ps timescale from an intermediate emerging after a ~500 fs vibrational cooling component. To glean further structural information from the data, we innovated a new 2D Raman approach for comprehensive visualization of anharmonically coupled vibrations. Using this method, we uncover a 232 cm^-1 out-of-plane bending mode modulating a 867 cm^-1 phenol hydrogen out-of-plane (HOOP) mode as HBDI evolves into the TICT state, providing compelling evidence for the functional relevance of conformational twisting as the molecule approaches the conical intersection.

The interplay between chromophore environment and fluorescence is further revealed by studying the effects of adding a viscous glycerol solvent. In contrast to that in pure water, HBDI in glycerol exhibits longer excited state dynamics, echoing the increased excited state lifetimes of the molecule within the protein pocket. Surprisingly, the 232 cm^-1 vibrational modulation disappears upon adding glycerol; instead, a 125 cm^-1 weak out-of-plane mode and a 275 cm^-1 in-plane methyl rocking mode appear. This change in structural dynamics is likely because the energy barrier for the 232 cm^-1 out-of-plane mode mode becomes prohibitively large in glycerol, whereas this is not the case for the two new modes. Differences in the vibrational coupling matrix due to a changing local environment show that increasing steric hindrance significantly inhibits the efficacy of nonradiative energy dissipation through the conical intersection for HBDI. In context of the GFP chromophore, the β-barrel of the protein pocket provides an intrinsic barrier to HBDI rotation that prevents conformational twisting as an alternative pathway to fluorescence. These findings reveal the molecular motions that govern the fate of the GFP chromophore in solution, and correlate the appearance of these specific motions with chromophore environment. New knowledge about the chromphoreâ??s photophysical properties provides valuable information for the quest to engineer better fluorescent proteins to benefit the growing bioimaging community.