EAPSI: Monitoring High Resolution Broadband Fluorescence of the Green Fluorescent Protein Model Chromophore

Taylor Miles A, Corvallis OR

Investigators

Abstract

The discovery of the Green Fluorescent Protein (GFP) has defined the way to visualize cellular events and life processes. By using GFP as a genetically encodable fluorescence reagent in super-resolution microscopy, non-invasive live cell imaging has powered numerous advances in life sciences. A mechanistic understanding of how the GFP chromophore works in the protein pocket, and why its fluorescence vanishes in solution, remains unclear. This knowledge gap hinders our understanding of important factors which lead to efficient emission. In collaboration with Dr. Justin Hodgkiss, an expert on the newly developed Transient Grating Photoluminescence Spectroscopy (TGPLS), research will be conducted at Victoria University of Wellington, New Zealand. This unique opportunity will allow the study of the broadband fluorescence signal of the GFP chromophore in solution on the intrinsic molecular timescale and will further help to characterize the fluorescent state of the chromophore within the protein. To design improved fluorescent proteins for high-resolution microscopy, a fundamental understanding of the hydrogen bonding network around a dynamic GFP chromophore is critical. Within the protein, the locked Ser-Tyr-Gly chromophore cannot isomerize and is inevitably forced to release energy through fluorescence. Outside the protein matrix, the interplay between fluorescence and isomerization drops the quantum yield from ~0.8 to ~2x10-4. This striking change of fluorescence indicates that the excited state population of 4-hydroxybenzylidene-1,2-dimethylimidazolinone (HBDI) travels through a conical intersection from S1 to S0, while the rest proceeds through fluorescence. Because of both the spatial and temporal resolution of TGPLS, it is a prime tool to study the time-resolved fluorescence profile of HBDI and gain further insights into the electronic excited state intermediates after photoexcitation. Collaboration with Dr. Hodgkiss will help delineate how fluorescence occurs from a small portion of the HBDI excited state and allow us to better understand the role the protein matrix plays in fluorescence. This award, under the East Asia and Pacific Summer Institutes program, supports summer research by a U.S. graduate student and is jointly funded by NSF and the Royal Society of New Zealand.