Resolving the ultrafast dynamics of the anionic green fluorescent protein chromophore in water

Abstract

The chromophore of the green fluorescent protein (GFP) is critical for probing environmental influences on fluorescent protein behavior. Using the aqueous system as a bridge between the unconfined vacuum system...

Photoswitchable Fluorescent Proteins: Mechanisms on Ultrafast Timescales

“…The exact twisting coordinates (dihedral angles, one-bond flip, or hula twist) and geometries and whether intermediate states are involved remain elusive for most RSFPs [ 36 , 37 , 38 ]. Studies on photoisomerization events have been a hot topic for the HBDI chromophore and its derivatives in solution, but the gained knowledge cannot be directly transferred into a chromophore in the protein matrix due to the complex electrostatic and steric interactions between the chromophore and surrounding protein residues, which are intrinsically more heterogeneous and asymmetric than the solution environment [ 39 , 40 , 41 , 42 ]. Therefore, it is urgent and necessary to elucidate the detailed structural motions of the chromophore and local amino acids in the electronically excited and “hot” (non-equilibrium) ground states, which are essential for the deepened understanding and rational development of next-generation RSFPs [ 43 ].…”

Fluorescence Modulation by Ultrafast Chromophore Twisting Events: Developing a Powerful Toolset for Fluorescent-Protein-Based Imaging

“…To capture the molecular twisting events of these embedded chromophores on their intrinsic time scales, suitable ultrafast techniques need to be carefully selected and implemented. Structural motions often lead to a change of charge distribution and dipole moment, particularly for the donor–acceptor compounds. , The specific two-ring system of the GFP chromophore (Figure ) represents a great example wherein the charge in the P-ring could be transferred to the I-ring following photoexcitation and the twisting motions along the methine bridge likely form a twisted intramolecular charge transfer (TICT) state with a reduced energy gap between the ground (S 0 ) and excited (S 1 ) states. ,, These twisting/torsional motions could significantly decrease the transition oscillator strength between S 1 and S 0 (forming a dark state), while a hot ground state (HGS) can quickly form out of the TICT state through a conical intersection (CI). , The conformational events can also lead to clear frequency changes of characteristic excited-state vibrational modes because of their high sensitivity to nonequilibrium structural changes with generally larger electric polarizabilities than their ground-state counterparts at thermal equilibrium, , which present an engaging and effective experimental opportunity to monitor and elucidate the underlying reaction coordinates of the photoexcited chromophore.…”

“…Such motions effectively depopulate the excited state, − suggesting that twisting is an intrinsic property of FP chromophores. This event has been investigated by many ultrafast spectroscopic techniques ,− and advanced calculation or simulation methods ,− from the gas phase to the condensed phase. However, most of these studies were focused on anionic p -HBDI and/or its derivatives in vacuo or in solution, and debates remain regarding the exact light-induced pathways, such as sequential or parallel roles played by P-ring and imidazolinone ring (I-ring) twisting motions (see Figure , left panel), and whether or not these processes are barrierless on ultrafast time scales .…”

“…This event has been investigated by many ultrafast spectroscopic techniques ,− and advanced calculation or simulation methods ,− from the gas phase to the condensed phase. However, most of these studies were focused on anionic p -HBDI and/or its derivatives in vacuo or in solution, and debates remain regarding the exact light-induced pathways, such as sequential or parallel roles played by P-ring and imidazolinone ring (I-ring) twisting motions (see Figure , left panel), and whether or not these processes are barrierless on ultrafast time scales . In contrast, our recent works have targeted and directly observed the onset, progression, and outcome of the chromophore twisting events inside the protein matrix in a series of FP derivatives and FP-based biosensors, ,− as well as photoconvertible and photoswitchable proteins …”

“…However, most of these studies were focused on anionic p-HBDI and/or its derivatives in vacuo or in solution, and debates remain regarding the exact light-induced pathways, such as sequential or parallel roles played by P-ring and imidazolinone ring (I-ring) twisting motions (see Figure 1, left panel), and whether or not these processes are barrierless on ultrafast time scales. 33 In contrast, our recent works have targeted and directly observed the onset, progression, and outcome of the chromophore twisting events inside the protein matrix in a series of FP derivatives and FP-based biosensors, 15,34−36 as well as photoconvertible and photoswitchable proteins. 37 Herein, from an experimental physical chemistry viewpoint, we aim to provide the essential electronic and vibrational signatures and kinetics of the twisted chromophore states of FPs, which are likely ubiquitous for photosensitive biomolecules with a light-sensing "active site" (chromophore) and inherent conformational flexibility.…”