Controlling Light-Induced Proton Transfer from the GFP Chromophore.

The front cover artwork is provided by the groups of Assoc. Prof. Anastasia V. Bochenkova (Lomonosov Moscow State University) and Prof. Lars H. Andersen (Aarhus University). The image shows the quantum nature of wavelength-dependent excited-state proton transfer in gas-phase H-bonded complexes of the GFP chromophore with an anionic proton acceptor. Read the full text of the Article at 10.1002/cphc.202100068.

Controlling Light-Induced Proton Transfer from the GFP Chromophore.

Green Fluorescent Protein (GFP) is known to undergo excited-state proton transfer (ESPT). Formation of a short H-bond favors ultrafast ESPT in GFP-like proteins, such as the GFP S65T/H148D mutant, but the detailed mechanism and its quantum nature remain to be resolved. Here we study in vacuo, light-induced proton transfer from the GFP chromophore in hydrogen-bonded complexes with two anionic proton acceptors, I- and deprotonated trichloroacetic acid (TCA- ). We address the role of the strong H-bond and the quantum mechanical proton-density distribution in the excited state, which determines the proton-transfer probability. Our study shows that chemical modifications to the molecular network drastically change the proton-transfer probability and it can become strongly wavelength dependent. The proton-transfer branching ratio is found to be 60 % for the TCA complex and 10 % for the iodide complex, being highly dependent on the photon energy in the latter case. Using high-level ab initio calculations, we show that light-induced proton transfer takes place in S1 , revealing intrinsic photoacid properties of the isolated GFP chromophore in strongly bound H-bonded complexes. ESPT is found to be very sensitive to the topography of the highly anharmonic potential in S1 , depending on the quantum-density distribution upon vibrational excitation. We also show that the S1 potential-energy surface, and hence excited-state proton transfer, can be controlled by altering the chromophore microenvironment.

Intrinsic photoisomerization dynamics of protonated Schiff-base retinal.

The retinal protonated Schiff-base (RPSB) in its all-trans form is found in bacterial rhodopsins, whereas visual rhodopsin proteins host 11-cis RPSB. In both cases, photoexcitation initiates fast isomerization of the retinal chromophore, leading to proton transport, storage of chemical energy or signaling. It is an unsolved problem, to which degree this is due to protein interactions or intrinsic RPSB quantum properties. Here, we report on time-resolved action-spectroscopy studies, which show, that upon photoexcitation, cis isomers of RPSB have an almost barrierless fast 400 fs decay, whereas all-trans isomers exhibit a barrier-controlled slow 3 ps decay. Moreover, formation of the 11-cis isomer is greatly favored for all-trans RPSB when isolated. The very fast photoresponse of visual photoreceptors is thus directly related to intrinsic retinal properties, whereas bacterial rhodopsins tune the excited state potential-energy surface to lower the barrier for particular double-bond isomerization, thus changing both the timescale and specificity of the photoisomerization.

Elucidation of the intrinsic optical properties of hydrogen-bonded and protonated flavin chromophores by photodissociation action spectroscopy.

A model system of the flavin chromophore was synthesized and investigated for its intrinsic optical properties by gas phase action spectroscopy using an ion storage ring. An ammonium group was anchored to this flavin chromophore to allow its transfer to the gas phase by electrospray ionization and for studying the influence of hydrogen bonding and a nearby positive charge. According to calculations one of the hydrogen atoms of the ammonium group favorably forms an intramolecular ionic hydrogen bond to one of the oxygen atoms of the flavin chromophore, and this interaction was found to cause a blueshift of the S0 → S1 transition and a redshift of the S0 → S2 transition. For comparison, the S0 → S1 transition shows little solvent dependence (only in regard to the degree of fine structure). In addition, the influence of protonation of the flavin chromophore was elucidated by experimental and theoretical studies of a simple flavin system. While the position of the S0 → S1 absorption was at identical positions in the gas phase for the intramolecularly hydrogen-bonded and protonated flavin systems, the S0 → S2 absorption was further redshifted for the protonated species. This redshift resulting from protonation was also observed in solution.

Action and Ion Mobility Spectroscopy of a Shortened Retinal Derivative.

The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are. Graphical Abstract.

The UV-visible action-absorption spectrum of all-trans and 11-cis protonated Schiff base retinal in the gas phase.

The UV-visible absorption of retinal in its protonated Schiff-base form is studied in the gas phase. In particular, transitions to highly-excited electronic states, Sn, in the all-trans and 11-cis forms are considered, and several new states are discovered. Their positions and strengths are compared to state of the art quantum calculations. The location of these states are particularly important when new fs pump-probe experiments are designed to investigate the fast excited-state dynamics of retinal chromophores.

Origin of the Intrinsic Fluorescence of the Green Fluorescent Protein.

Green fluorescent protein, GFP, has revolutionized biology, due to its use in bioimaging. It is widely accepted that the protein environment makes its chromophore fluoresce, whereas the fluorescence is completely lost when the native chromophore is taken out of GFP. By the use of a new femtosecond pump-probe scheme, based on time-resolved action spectroscopy, we demonstrate that the isolated deprotonated GFP chromophore can be trapped in the first excited state when cooled to 100 K. The trapping is shown to last for 1.2 ns, which is long enough to establish conditions for fluorescence and consistent with calculated trapping barriers in the electronically excited state. Thus, GFP fluorescence is traced back to an intrinsic chromophore property, and by improving excited-state trapping, protein interactions enhance the molecular fluorescence.

Decoupling Electronic versus Nuclear Photoresponse of Isolated Green Fluorescent Protein Chromophores Using Short Laser Pulses.

The photophysics of a deprotonated model chromophore for the green fluorescent protein is studied by femtosecond laser pulses in an electrostatic ion-storage ring. The laser-pulse duration is much shorter than the time for internal conversion, and, hence, contributions from sequential multiphoton absorption, typically encountered with ns-laser pulses, are avoided. Following single-photon excitation, the action-absorption maximum is shown to be shifted within the S_{0} to S_{1} band from its origin at about 490 to 450 nm, which is explained by the different photophysics involved in the detected action.

A PYP chromophore acts as a 'photoacid' in an isolated hydrogen bonded complex.

The light-induced response of a neutral Photoactive Yellow Protein chromophore in a hydrogen-bonded complex with a proton acceptor has been studied by dual-detection action absorption spectroscopy and density functional theory. We show that the chromophore is a 'photoacid' and that ultrafast excited-state proton transfer might be operative in an isolated complex.

Analysis of ionic photofragments stored in an electrostatic storage ring.

A new method to analyze the properties of fragment ions created in storage ring experiments is presented. The technique relies on an acceleration of ionic fragments immediately after production whereby the fragments are stored in the storage ring. To obtain a fragment mass spectrum, the storage ring is exploited as an electrostatic analyzer (ESA) in which case the number of stored fragment ions is recorded as a function of the applied acceleration potential. However, the storage ring can additionally be employed as a time-of-flight (TOF) instrument by registering the temporal distribution of fragment ions. It is demonstrated that the combined ESA-TOF operation of the ring allows not only to determine fragment masses with much better resolution compared to the ESA mode alone but also enables the extraction of detailed information on the fragmentation dynamics. The method is described analytically and verified with photodissociation experiments on stored Cl2 (-) at an excitation wavelength of 530 nm.

How far can a single hydrogen bond tune the spectral properties of the GFP chromophore?

Photoabsorption of the hydrogen-bonded complex of a neutral and an anionic Green Fluorescent Protein chromophore has been studied using a new dual-detection approach to action-absorption spectroscopy. Following absorption of one photon, dissociation through a single channel ensures that the full absorption spectrum is measured. Our theoretical account of the spectral shape reveals that the anionic 0-0 transition (464 nm) is blue-shifted compared to that of the wild-type protein (478 nm) due to the stronger H-bond in the dimer, and represents an upper bound for that of the isolated anion. At the same time, the apparent effect of the H-bond for the neutral chromophore is as large as 0.5 eV, red-shifting the absorption maximum of the isolated neutral (340 nm) to that measured in the dimer (393 nm) and various proteins (∼395 nm). This shift results from changes in the topography of potential-energy surfaces in the Franck-Condon region of the H-bonded systems.