Green fluorescent protein (GFP): low-frequency vibrations switch on the light

The fluorescence of green fluorescent protein (GFP) arises from an excited-state proton transfer (ESPT), in which a light-absorbing chromophore relays its phenolic proton along a hydrogen-bonded chain through a water molecule and Ser205 to Glu222; the reaction occurs in a concerted fashion within about one picosecond (ps) A team at the University of Naples in Italy combined excited-state ab initio molecular dynamics (AIMD) with wavelet analysis to track this process simultaneously in the time and frequency domains, identifying the main driving force that opens the reaction as a relaxation in which the twisting motion of the chromophore's rings softens (shifts to lower frequency) upon absorbing light Activated low-frequency vibrations in the roughly 100–120 cm⁻¹ range tighten the hydrogen-bonded chain and open the path for the proton to travel, and these simulation results matched earlier femtosecond stimulated Raman spectroscopy (FSRS) experiments closely [Quantum Biology Society] Green fluorescent protein (GFP), derived from a jellyfish, has become an indispensable labelling tool in modern life-science imaging. The secret of its green glow lies in a remarkable little drama of photochemistry. When the chromophore, having absorbed blue light, releases the proton (H⁺) on its phenol group, that proton is relayed along a hydrogen-bonded chain through a crystallographic water molecule and the Ser205 residue to the final acceptor, Glu222. The anionic form left behind then radiates green fluorescence at around 508 nm. This excited-state proton transfer (excited state proton transfer, ESPT) is one of the simplest and best-studied cases of a biological proton shuttle. Yet what molecular-level motion actually throws the door of this relay open in the instant just after light is absorbed has long been fiercely debated. In particular, an experimental proposal that a low-frequency wobbling motion of the chromophore triggers the reaction has stood against a counterargument that questions it. A team led by Greta Donati and Professor Nadia Rega of the University of Naples Federico II (Università di Napoli Federico II) took this debate head-on in a paper published in Chemical Science (Royal Society of Chemistry), volume 9, in 2018. The researchers simulated the reaction directly with excited-state ab initio molecular dynamics (AIMD) and dissected the resulting trajectories with a multi-resolution wavelet analysis. They found that the main driving force opening the reaction is the softening (shift toward lower-frequency bands) of the twisting motion between the chromophore's two rings upon absorbing light, and that this relaxation tightens the hydrogen-bonded chain to clear a path for the proton. They further showed that the temporal evolution of the vibrations their simulation produced lines up excellently with the latest femtosecond Raman experiments. ■ Before the light comes on: the moment the chromophore loosens up In the ground state the chromophore prefers a flat structure in which its two rings lie almost in one plane (the N–C–C–C dihedral angle that measures the ring twist averages about −2 degrees), and the O–H bonds that would donate a proton sit in firm covalent states, so there is no physical reason for the reaction to occur. Under physiological conditions the chromophore exists with the neutral (protonated) form outnumbering the anionic form by about sixfold. But once it absorbs blue light and enters the excited state, the situation changes abruptly. In as little as 15–20 femtoseconds (fs) the oxygen–oxygen (O–O) distances within the hydrogen-bonded chain begin to close and the O–H bonds loosen. The chromophore's two rings start to twist out of plane, opening up to as much as 20–30 degrees. In the first trajectory analysed, the proton transfer completed in just 720 fs after all of this, with the three successive proton movements occurring in a concerted fashion almost without lag. In the remaining, randomly selected trajectories the reaction also finished successfully within one picosecond (ps) — between 350 and 980 fs. ■ The key: low-frequency vibrations in the ~100–120 cm⁻¹ range The researchers examined the trajectories in depth with wavelet analysis. Wavelet analysis is an advanced technique that visualizes when a particular vibration is activated along both the time and frequency axes at once, drawing a kind of score for the molecular vibrations as they fluctuate moment to moment. The core finding it revealed is that at the instant light is absorbed, certain low-frequency collective vibrations soften dramatically (their frequencies drop). Normal-mode analysis showed that the vibration twisting the chromophore's rings fell from 146 cm⁻¹ to 101 cm⁻¹, and the vibration in which the phenol ring's out-of-plane O–H and the chain oxygens move together fell from 172 cm⁻¹ to 117 cm⁻¹. The dihedral angle representing the overall planarity of the hydrogen-bonded chain likewise swung strongly with a period of about 250–300 fs (about 110–130 cm⁻¹). The researchers interpreted this softening as newly granting the chromophore the freedom to twist its rings, and that loosened play pulling the hydrogen-bonded chain into alignment, drawing the proton's path taut. By analogy, it is like a stiff joint that must first loosen before the hand can reach far enough to pass the baton. ■ Where simulation meets femtosecond Raman At this juncture the paper casts a weighty vote in a long-running scholarly debate. Earlier, on the basis of femtosecond stimulated Raman spectroscopy (FSRS) results, Mathies and coworkers had proposed that a roughly 120 cm⁻¹ low-frequency wagging of the light-struck chromophore's phenol ring directly promotes the reaction. That figure agrees strikingly with the oxygen-involving low-frequency mode (about 120 cm⁻¹) that this simulation found to modulate the relaxation of the chromophore and its pocket. Some researchers had questioned this causal link. But in this study the wavelet spectra for the chromophore's C–O and C–N bond distances showed a clear pattern of oscillating out of phase with each other on a period of about 250 fs, in full accord with the distinctive behaviour that past FSRS experiments had recorded. On this basis the researchers concluded that the process of tension unwinding inside the chromophore is not a simple, monotonic one-dimensional progression but takes on a dynamic oscillatory character that rises and falls in step with the low-frequency vibrations. Because the simulation reproduced the experiment's peculiar Raman signals while tracking the excited-state electronic relaxation, it became possible at last to decode what those experimental signals actually mean at the molecular level. ■ A supporting player, His148, and a careful self-assessment The His148 residue, which forms a hydrogen bond with the chromophore's phenol ring, is a dependable supporting player in this photochemical drama. About 200 fs before the proton transfer, His148 was seen to move sharply toward the chromophore (the distance between its hydrogen and the phenolic oxygen contracting below 2 Å), gripping and stabilizing the freshly formed anion. The researchers also set out their study's limitations transparently. The reaction speed in the simulation (about 720 fs) is somewhat faster than the several-picosecond scale observed in actual experiments, which they attribute to the density functional theory (DFT) model underestimating the reaction energy barrier and omitting the polarization effects of the molecular-mechanics (MM) region residues, His148 among them. They also made clear that this dynamics model rests on the Born–Oppenheimer approximation, which treats the atomic nuclei classically. The most reliable insight the study offers, therefore, is not the absolute reaction speed itself but the choreography of the vibrations that open the reaction's door and the timing of that mechanism — and it is precisely here that it achieved striking harmony with the experimental data. ■ Significance and outlook This paper's greatest novelty lies in its methodology. By combining excited-state ab initio dynamics (AIMD) with wavelet analysis, it directly compared photoactivated vibrations with femtosecond Raman experiments for the first time and gave a clear reading of the role played by low-frequency modes. Through this the researchers demonstrated that ab initio simulation can become a powerful, routine tool for interpreting the data of difficult, cutting-edge time-resolved spectroscopy experiments. From the standpoint of quantum biology, GFP's proton chain is an excellent reference model for probing the mechanisms of biological proton transfer. This study did not directly compute proton tunneling or nuclear quantum effects as such, but by vividly visualizing, through classical nuclear dynamics, the role of the vibrational modes that unbar the reaction pathway, it captured the essence of the remarkable molecular mechanics by which living things handle light. #QuantumBiology #GreenFluorescentProtein #GFP #ExcitedStateProtonTransfer #ESPT #ProtonWire #AbInitioMolecularDynamics #WaveletAnalysis #FemtosecondRaman #Photochemistry https://pmc.ncbi.nlm.nih.gov/articles/PMC5890789/