Photoinduced proton transfer inside an engineered green fluorescent protein: a stepwise–concerted-hybrid reaction

Abstract

Photoactivated proton transfer (PT) wire is responsible for the glow of green fluorescent protein (GFP), which is crucial for bioimaging and biomedicine. In this work, a new GFP-S65T/S205V double mutant is developed from wild-type GFP in which the PT wire is significantly modified. We implement femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS) to delineate the PT process in action. The excited state proton transfer proceeds on the ∼110 ps timescale, which infers that the distance of one key link (water to T203) in the PT wire of GFP-S205V is shortened by the extra S65T mutation. The rise of an imidazolinone ring deformation mode at ∼871 cm-1 in FSRS further suggests that this PT reaction is in a concerted manner. A ∼4 ps component prior to large-scale proton dissociation through the PT wire is also retrieved, indicative of some small-scale proton motions and heavy-atom rearrangement in the vicinity of the chromophore. Our work provides deep insights into the novel hybrid PT mechanism in engineered GFP and demonstrates the power of tunable FSRS methodology in tracking ultrafast photoreactions with the desirable structural specificity in physiological environments.

Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman Spectroscopy

“…The kinetic model for transient molecular species and the match between experimental and fitted traces across the spectral detection window at four representative time delay points are shown in Figure S1 (see the Supplementary Materials ). Previous studies on related proteins have shown that a purely concerted model does not fit the TA spectra well, and some preparation stages typically exist before the main ESPT reaction step [ 21 , 26 ]. The resultant evolution-associated spectra (EAS) are plotted in Figure 2 b with a root-mean-square (RMS) value of 1.318 × 10 −3 .…”

“…The small emission peak at 512 nm is likely from the fluorescence background (because it is present to a certain extent at negative time delay points) or some ultrafast ESPT channel via those pre-existing, largely optimal H-bonding chains [ 29 , 32 , 45 ]. The initial EAS then evolves to the second EAS with a time constant of 3.8 ps, during which the intensity of the A* SE band near 478 nm increases, indicating the necessity of a preparation stage before the ESPT process [ 4 , 21 , 26 , 46 ]. The ESA band below 450 nm also blue shifts, suggesting that the A* S 1 state is stabilized on this time scale.…”

“…Judging from the TA profile of A* species before the main ESPT step, the initial two EAS can be attributed to a well-defined A* state with a notable slope out of the Franck–Condon (FC) region, wherein the chromophore remains protonated. Reminiscent of a previously proposed stepwise-concerted-hybrid ESPT reaction mechanism in a GFP-S65T/S205V double mutant [ 26 ], the initial ~3.8 ps process likely involves some small-scale proton motions in close proximity to the chromophore (e.g., CRO···water, see Figure 1 a) and a charge transfer (CT) step [ 26 , 32 , 47 ] in the partially deprotonated chromophore, which presumably set up the stage for the subsequent main ESPT step on the hundreds of ps time scale (see below).…”

“…The essence of performing time-resolved FSRS is to capture transient Raman modes in the excited state before the system returns to the electronic ground state [ 7 , 10 ], so the resonance conditions of the Raman pump–probe pair need to be carefully selected to avoid generating significant ground and excited state species simultaneously (which could lead to spectral overlap and dispersive line shapes [ 31 , 52 ]). Fortunately, pre-resonance conditions have been experimentally validated to track excited state vibrational dynamics during a photochemical reaction, such as ESPT in solution [ 12 , 32 ] or protein environment [ 8 , 11 , 13 , 23 , 26 ], also showing apparent vibrational frequency shifts from the ground to excited state (e.g., S 0 →S 1 ) of the chromophore.…”

“…FSRS has been successfully implemented to investigate a wide array of photosensitive systems, such as rhodopsins [ 3 , 14 , 15 ], phytochrome [ 16 ], myoglobin [ 17 ], blue-light photoreceptors using flavin [ 18 , 19 ], fluorescent proteins (FPs), and FP-based biosensors [ 4 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ], photomolecular rotors [ 27 , 28 ], and photoacids [ 12 , 29 , 30 , 31 , 32 ]. Among them, wild-type green fluorescent protein (wtGFP) is a prototypical molecular machine [ 33 , 34 ].…”